WO2017079983A1

WO2017079983A1 - USES OF MICRORNA MIR-574-5p-BASED COMPOUNDS AS IMMUNOMODULATORS AND COMPOSITIONS THEREOF

Info

Publication number: WO2017079983A1
Application number: PCT/CN2015/094617
Authority: WO
Inventors: James Y. Yang; Tao Wang; Yu LUO; Jianchun CAI; Feng Zheng
Original assignee: Amoigen Bioscience (Xiamen) Company Limited
Priority date: 2015-11-13
Filing date: 2015-11-13
Publication date: 2017-05-18
Also published as: CN107206104A

Abstract

Provided are the preventive or therapeutic uses of miR-574-5p, miR-574-5p mimics or derivatives, and miR-574-5p inhibitors as immune modulators, which exert actions through modulating TLR7 and/or TLR8 signaling. Also provided are compositions containing miR-574-5p, miR-574-5p mimics or derivatives, or miR-574-5p inhibitors, and administration methods thereof.

Description

USES OF MICRORNA MIR-574-5p-BASED COMPOUNDS AS IMMUNOMODULATORS AND COMPOSITIONS THEREOF

FIELD OF THE INVENTION

The present invention relates generally to the fields of immunomodulation and immunotherapy. More specifically, the invention relates to the uses of miR-574-5p, miR-574-5p mimics or derivatives, and miR-574-5p inhibitors, especially uses as immunomodulators and/or adjuvants, preferably through modulating mammalian TLR7 and/or mammalian TLR8 signaling. The invention also relates to compositions containing miR-574-5p, miR-574-5p mimics or derivatives, or miR-574-5p inhibitors as immunomodulators and/or adjuvants. The invention further relates to methods for treating or preventing diseases or disorders related to aberrant mammalian TLR7 and/or mammalian TLR8 signaling comprising of administering miR-574-5p, miR-574-5p mimics or derivatives, or miR-574-5p inhibitors, or the compositions containing miR-574-5p, miR-574-5p mimics or derivatives, or miR-574-5p inhibitors to subjects in need thereof.

BACKGROUND

In response to pathogenic insults, an organism first mounts an innate immune response, employing a variety of transmembrane and secreted molecules, followed by the activation of adaptive immune response, as well as the induction of inflammation mediators (Ciraci et al., 2012) . Pattern recognition receptors (PRRs) such as the Toll-like receptors (TLRs) family proteins have emerged as important components of the innate immune response, recognizing several different generic pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) , and thus eliciting a rapid immune response (Cervantes et al., 2012, Aderem and Ulevitch, 2000, Kawai and Akira, 2009, Kawai and Akira, 2011, Akira et al., 2001, Pandey and Agrawal, 2006, Kawai and Akira, 2007a) . As type-I transmembrane proteins, TLRs are characterized by an extracellular domain composing of leucine-rich repeats and a cytoplasmic domain that is homologous with the mammalian type I receptor for interleukin 1 (IL-1R) , termed Toll/interleukin-1 receptor domain (TIR) (Gay and Keith, 1991) . The ectodomain of TLRs is responsible for the recognition of PAMPs and DAMPs, while the cytoplasmic domain is required for downstream signaling (Takeda and Akira, 2005) . So far, 13 TLRs (designated TLR1 to TLR13) have been described in humans or mice (O'Neill et al., 2013) , with their expression patterns highly selective and contingent upon their PAMP/DAMP recognition properties (Chuang and Ulevitch, 2000, Du et al., 2000, Rock et al., 1998, Takeuchi et al., 1999, Chuang and Ulevitch, 2001, Barton and Medzhitov, 2003, Hedayat et al., 2012) .

A number of studies have shown that TLRs evoke immune and inflammatory response through endogenous and exogenous ligands recognition (Akira and Takeda, 2004, Moghimpour Bijani et al., 2012, Akira et al., 2001) , for which reason they are localized differently, i.e., on the cell surface or in endosomal compartments (Kawai and Akira, 2007b, Latz et al., 2004, Oldenburg et al., 2012) . TLR7 and TLR8 are highly homologous, and together with TLR3 and TLR9 form a subgroup within the TLR superfamily, as they all recognize nucleic acids, and are expressed in endosomes and require endosomal maturation for signaling (Du et al., 2000, Liu et al., 2010, Heil et al., 2003) . Although viral ssRNAs and synthetic ssRNAs were previously thought to be the only ligands for TLR7 and TLR8 (Heil et al., 2004, Diebold et al., 2004, Lund et al., 2004) , small molecules such as imidazoquinolines and certain nucleoside analogues have been shown to activate TLR7 and TLR8 and to induce TLR signaling (Lee et al., 2003, Schon and Schon, 2008, Hemmi et al., 2002, Jurk et al., 2002) . In humans, TLR7 is mainly expressed in plasmacytoid dendritic cells (pDC) , B-cells, and neutrophils, whereas TLR8 is highly expressed in monocytes, macrophages, myeloid dendritic cells (mDC) and neutrophils (Chuang and Ulevitch, 2000, Iwasaki and Medzhitov, 2004, Hemmi et al., 2002, Jurk et al., 2002, Gorden et al., 2005, Jurk et al., 2006) . As a result of these differences in cellular expression, activation of the immune response through human TLR7 leads to a response dominated by Type I IFN production, whiles activation through human TLR8 induces multiple pro-inflammatory cytokines such as TNF, IL-12, IL-6, IL-8 and IL-1 (Barrat et al., 2005, Gorden et al., 2005, Pasare and Medzhitov, 2005) . Therefore, activation of the immune response through TLR7 and TLR8 mainly leads to induction of T-helper 1 (Th1) -type immune responses (Pasare and Medzhitov, 2005) . Identification of novel and efficient TLR7 and/or TLR8 agonists and/or antagonists is of significant importance in therapy of inflammatory disorders, autoimmune diseases, and cancers.

MicroRNAs (miRNAs) , which are small non-coding RNAs (18 to 25 nucleotides in length) , normally regulate gene expression by binding to the 3’ -untranslated region (3’ UTR) of the messenger RNA (mRNA) targets to induce mRNA degradation and to inhibit translation (Bartel, 2009, Bartel, 2004, Ambros, 2004) . As important regulators of gene expression, miRNAs have been implicated in many biological processes and pathological conditions including cell proliferation, differentiation and apoptosis, neuroprocesses, carcinogenesis, immune response, viral pathogenesis, and metabolic disease (Miska, 2005, Kutay et al., 2006, Visone et al., 2008, Saba et al., 2014, Jiang et al., 2014, Hammond, 2015) . More recently, the pivotal role of miRNAs in immune response has been shown, whereby miRNAs are implicated in the regulation of B and T cells development and differentiation, proliferation of monocytes and neutrophils, antibody switching and the release of inflammatory mediators (Lindsay, 2008) . Conversely, dysregulated expression of miRNAs has been reported in various disease conditions such as cancer, autoimmune disease, cardiovascular disease, and neurodegenerative disease (Bushati and Cohen, 2007, Chang and Mendell, 2007) .

Several studies have shown that a range of miRNAs are involved in various aspects in the regulation of the immune and inflammatory response (Li et al., 2007, Rodriguez et al., 2007, Fontana et al., 2007, Johnnidis et al., 2008, Taganov et al., 2006, Hou et al., 2009, O'Connell et al., 2007, Tili et al., 2007, Sheedy et al., 2010) . A number of recent studies have shown that some miRNAs directly or indirectly act as important modulators of the TLR signaling pathway (Tserel et al., 2011) (Olivieri et al., 2013) (Bai et al., 2012, Sun et al., 2012, Alam and O'Neill, 2011) , which therefore helps in controlling and fine-tuning the immune response as well as the development of immune-related diseases (O'Neill et al., 2011) . For TLR7 and TLR8, a number of natural and synthetic immunostimulatory ligands have been described (reviewed in (Smits et al., 2008) ) . Moreover, as short ssRNA molecules, miRNAs were suggested to mimic viral RNA and consequently bind directly to TLR7 and/or TLR8, thus leading to the detection of pathogenic nucleic acids (Olivieri et al., 2013) . MiRNAs can therefore function as agonists of the ssRNA-binding TLRs, leading to NF-κB signaling activation and secretion of pro-inflammatory cytokines (Fabbri, 2012) . Thus, given the central role that miRNAs seem to play in immune response, it is conceivable that they are currently being leveraged for therapeutic purposes (Van & Kauppinen, 2014) . In particular, it is of great interest in the application of miRNA ligands/agonists/antagonists for TLRs in therapeutics, especially in immunotherapeutics and as vaccine adjuvants (Chew and Abastado, 2013, Rhee et al., 2010, Hennessy et al., 2010) . This premise forms the basis of this disclosure.

The human miRNA gene MIR574, which encode miR-574-5p (the miRNA of interest in this disclosure) and miR-574-3p, is located in the first intron region of the Fam114a1 gene on human Chromosome 4p14 locus, and is the direct neighbor of human TLR1/6/10, whereas mouse Mir574 gene is located on the Chromosome 5qC31 locus and is also in direct neighborhood with mouse Tlr1/6. MiR-574-5p is evolutionarily conserved among mammals and is highly rich in guanosine and uridine (GU-rich) . The expression of miR-574-5p is found in a broad range of tissues (Zhang et al., 2014) . A few studies have shown that abnormal overexpression of miR-574-5p is associated with various human cancers, while other studies suggested that this miRNA might serve as a biomarker for conditions such as sepsis and systemic lupus erythematosus (Meyers-Needham et al., 2012, Ranade et al., 2010, Ji et al., 2013, Mao et al., 2010) (Wang et al., 2012) . However, there is currently a dearth of information on the roles of miR-574-5p in the immune and inflammatory response and in the development of immune-related disease conditions.

SUMMARY OF THE INVENTION

The inventors recently found that miR-574-5p or miR-574-5p mimics or derivatives can serve as ligands to bind to mammalian TLR7 and/or mammalian TLR8, and thus triggering TLR7 and/or TLR8 mediated immune responses in vitro and in vivo. Therefore, miR-574-5p, miR-574-5p mimics or derivatives, or miR-574-5p inhibitors are useful in a number of applications related to TLR7 and/or TLR8 mediated immune and inflammatory responses, such as developing methods for treating or preventing conditions involving unwanted immune activity, such as inflammatory disorders, autoimmune diseases, and cancers. The inventors found the unique and broad-spectrum immunomodulatory capability of miR-574-5p, miR-574-5p mimics or derivatives, or miR-574-5p inhibitors, as ligands/agonists/antagonists for mammalian TLR7 and/or mammalian TLR8, which can be used to modulate the immune response for immunotherapy applications.

In a first aspect of the present invention, it provides the use of miR-574-5p, miR-574-5p mimics or derivatives, or combinations thereof as agonists for mammalian TLR7 and/or mammalian TLR8.

In one embodiment of the first aspect, the miR-574-5p mimics or derivatives are selected from the group consisting of phosphorothiolated (PS) -miR-574-5p, morpholino-miR-574-5p, 2’ -O-methyl-miR-574-5p, 2’ -O-methoxyethyl-miR-574-5p, 2’ -fluoro-miR-574-5p, or combinations thereof.

In another embodiment of the first aspect, it provides a composition for inducing mammalian TLR7 and/or mammalian TLR8 mediated immune responses in cells or a subject, containing miR-574-5p, miR-574-5p mimics or derivatives or combinations thereof, and/or pharmaceutically acceptable excipients.

In a further embodiment of the first aspect, it provides methods for inducing mammalian TLR7 and/or mammalian TLR8 mediated immune responses in a subject, comprising administering effective amount of miR-574-5p, miR-574-5p mimics or derivatives, or combinations thereof to a subject in need thereof.

In a still further embodiment of the first aspect, it provides methods for inducing mammalian TLR7 and/or mammalian TLR8 mediated immune responses in a subject, comprising administering effective amount of composition containing miR-574-5p, miR-574-5p mimics or derivatives, or combinations thereof to a subject in need thereof.

In a yet further embodiment of the first aspect, the mammalian TLR7 is mouse TLR7 (mTLR7) , and the mammalian TLR8 is human TLR8 (hTLR8) .

In a second aspect of the present invention, it provides the use of miR-574-5p inhibitors as antagonists for mammalian TLR7 and/or mammalian TLR8.

In one embodiment of the second aspect, the miR-574-5p inhibitors are selected from the group consisting of short hairpin RNA (shRNA) against miR-574-5p, single-stranded RNA complementary to miR-574-5p, double-stranded small interfering RNA (siRNA) targeting miR-574-5p, single-stranded DNA complementary to miR-574-5p； the mimics or derivatives of the single-stranded RNA complementary to miR-574-5p, double-stranded siRNA targeting miR-574-5p, and single-stranded DNA complementary to miR-574-5p in the forms of phosphorothiolate modification, morpholino modification, 2’ -O-methyl-modification, 2’ -O-methoxyethyl-modification, 2’ -fluoro-modification, locked nucleic acid (LNA) -modification； or combinations thereof.

In another embodiment of the second aspect, it provides a composition for inhibiting mammalian TLR7 and/or mammalian TLR8 mediated immune responses in cells or a subject, containing the miR-574-5p inhibitors and/or pharmaceutically acceptable excipients.

In a further embodiment of the second aspect, it provides a composition for inhibiting mammalian TLR7 and/or mammalian TLR8 mediated immune responses in cells or a subject, containing shRNA against miR-574-5p, single-stranded RNA complementary to miR-574-5p, double-stranded siRNA targeting miR-574-5p, single-stranded DNA complementary to miR-574-5p； the derivatives of the single-stranded RNA complementary to miR-574-5p, double-stranded siRNA targeting miR-574-5p, and single-stranded DNA complementary to miR-574-5p in the forms of phosphorothiolate modification, morpholino modification, 2’ -O-methyl-modification, 2’ -O-methoxyethyl-modification, 2’ -fluoro-modification, LNA-modification； or combinations thereof and/or pharmaceutically acceptable excipients.

In a still further embodiment of the second aspect, it provides methods for inhibiting mammalian TLR7 and/or mammalian TLR8 mediated immune responses in a subject, comprising administering effective amount of miR-574-5p inhibitors to a subject in need thereof.

In a yet further embodiment of the second aspect, it provides methods for inhibiting mammalian TLR7 and/or mammalian TLR8 mediated immune responses in a subject, comprising administering effective amount of shRNA against miR-574-5p, single-stranded RNA complementary to miR-574-5p, double-stranded siRNA targeting miR-574-5p, single-stranded DNA complementary to miR-574-5p； the derivatives of the single-stranded RNA complementary to miR-574-5p, double-stranded siRNA targeting miR-574-5p, and single-stranded DNA complementary to miR-574-5p in the forms of phosphorothiolate modification, morpholino modification, 2’ -O-methyl-modification, 2’ -O-methoxyethyl-modification, 2’ -fluoro-modification, LNA-modification； or combinations thereof to a subject in need thereof.

In a yet further embodiment of the second aspect, it provides methods for inhibiting mammalian TLR7 and/or mammalian TLR8 mediated immune responses in a subject, comprising administering effective amount of composition containing miR-574-5p inhibitors to a subject in need thereof.

In a yet further embodiment of the second aspect, the mammalian TLR7 is mouse TLR7 (mTLR7) , and the mammalian TLR8 is human TLR8 (hTLR8) .

In a third aspect of the present invention, it provides the use of miR-574-5p, miR-574-5p derivatives, or combinations thereof as adjuvants.

In one embodiment of the third aspect, the adjuvant can be administered together with a vaccine, an antibacterial agent, or an antigen to a subject in need thereof.

In another embodiment of the third aspect, it provides a composition containing miR-574-5p, miR-574-5p derivatives, or combinations thereof, and a vaccine or an antibacterial agent, as well as pharmaceutically acceptable excipients.

In a further embodiment of the third aspect, the miR-574-5p derivatives are selected from the group consisting of PS-miR-574-5p, morpholino-miR-574-5p, 2’ -O-methyl-miR-574-5p, 2’ -O-methoxyethyl-miR-574-5p, 2’ -fluoro-miR-574-5p, or combinations thereof.

In a still further embodiment of the third aspect, the mammalian TLR7 is mouse TLR7 (mTLR7) , and the mammalian TLR8 is human TLR8 (hTLR8) .

In a fourth aspect, the invention provides a method for treating a subject having diseases or disorders related to abnormal mammalian TLR7 and/or mammalian TLR8 signaling, for example cancer, autoimmune disorders, airway inflammation, inflammatory disorders, infectious diseases, skin disorders, allergy, asthma or diseases caused by pathogens, such method comprising administering to the patients having such disorders or diseases the miR-574-5p-based compound (s) according to the invention in a therapeutically effective amount.

In a fifth aspect, the invention provides a method for preventing diseases or disorders related to mammalian TLR7 and/or mammalian TLR8 signaling in a subject, for example cancer, an autoimmune disorder, airway inflammation, inflammatory disorders, infectious disease, skin disorders, allergy, asthma or diseases caused by a pathogen, such method comprising administering to a subject that is susceptible to such disorders or diseases the miR-574-5p-based compound (s) according to the invention in a therapeutically effective amount.

In certain embodiments of the present invention, the miR-574-5p, miR-574-5p derivatives, or miR-574-5p inhibitors can be used for preventing or treating the following diseases: autoimmune diseases and inflammatory disorders such as SLE, MS, and asthma； viral infection such as H5N1, VSV, and SARS； bacterial infection such as sepsis； graft versus host diseases； cancer such as lung cancer, pancreatic cancer, CRC, prostate cancer, and HNC； and cardiovascular and pulmonary arterial hypertension.

In a sixth aspect, the embodiments of the present invention provide methods of diagnosing risks related to immune-related conditions in a subject, comprising: (i) identifying the relative miR-574-5p expression compared to control, and (ii) diagnosing increased risk of immune related conditions in the subject if the subject has increased miR-574-5p expression compared to control, or (iii) diagnosing no increased risk of immune related conditions in the subject if the subject does not have increased miR-574-5p expression compared to control.

In a seventh aspect, the embodiments of the present invention provide use of miR-574-5p, miR-574-5p derivatives, or miR-574-5p inhibitors in the preparation of drugs for treating diseases or disorders related to mammalian TLR7 and/or mammalian TLR8 signaling.

In one embodiment of the seventh aspect, the miR-574-5p derivatives are selected from the group consisting of PS-miR-574-5p, morpholino-miR-574-5p, 2’ -O-methyl-miR-574-5p, 2’ -O-methoxyethyl-miR-574-5p, 2’ -fluoro-miR-574-5p, and combinations thereof, and wherein the miR-574-5p inhibitors is selected from the group consisting of shRNA against miR-574-5p, single-stranded RNA complementary to miR-574-5p, double-stranded siRNA targeting miR-574-5p, single-stranded DNA complementary to miR-574-5p； the derivatives of the single-stranded RNA complementary to miR-574-5p, double-stranded siRNA targeting miR-574-5p, and single-stranded DNA complementary to miR-574-5p in the forms of phosphorothiolate modification, morpholino modification, 2’ -O-methyl-modification, 2’ -O-methoxyethyl-modification, 2’ -fluoro-modification, LNA-modification； or combinations thereof.

DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the genomic locations of human MIR574 and mouse Mir574 genes, sequence conservation of miR-574-5p in mammals and sequence similarity between mammalian miR-574-5ps and virus-derived ssRNAs. FIG. 1a shows that human MIR574 gene is located on human Chromosome 4p. 14 and is the direct neighbor of hTLR1/6/10. FIG. 1b shows that mouse Mir574 gene is located on mouse Chromosome 5q. C31 and is the direct neighbor of mTLR1/6. FIG. 1c shows sequence alignment among mammals. bta, Bos taurus (cow) ； cfa, Canis familiaris (dog) ； efu, Eptesicus fuscus (big brown bat) ； ggo, Gorilla gorilla (gorilla) ； hsa, Homo sapiens (human) ； mmu, Mus musculus (mouse) ； ssc, Sus scrofa (pig) . FIG. 1d shows similarity between mammalian miR-574-5p miRNA sequences and three virus-derived ssRNA sequences.

FIG. 2 illustrates the RNA-protein interaction between miR-574-5p and mTlr7 or hTLR8 as determined by RNA-protein co-imunoprecipitation (co-IP) and the localization of miR-574-5p in endosome or lysosome compartment as determined by confocal fluorescence microscopy. FIG. 2a shows co-IP assays demonstrating the binding of Dig-miR-574-5p with a truncated hTLR8 but not a truncated hTLR7. FIG. 2b shows co-IP assays demonstrating the binding of Dig-miR-574-5p with the full-length hTLR8 but not the full-length hTLR7 and hTLR9. FIG. 2c shows co-IP assays demonstrating that the binding of Dig-miR-574-5p with a truncated mTLR7 but not a truncated mTLR8. FIG. 2d shows co-IP assays demonstrating the binding of Dig-miR-574-5p with the full-length mTLR7 but not the full-length mTLR8 or mTLR9. FIG. 2e shows confocal microscopy demonstrating localization of miR-574-5p in the endosome or lysosome compartment. HeLa cells were transfected with Dotap-conjugated Cy3-miR-574-5p (red) and stained with LysoTracker DND-22 (blue) and visualized under a confocal microscope.

FIG. 3 illustrates miRNA qPCR analyses of miR-574-5p overexpression or knockdown in human monocytic THP1 cells and cervical cancer HeLa cells. FIG. 3a shows the verification of miR-574-5p overexpression or knockdown in THP1 cells. ***, p < 0.001； pFlag-CMV2 transfected versus phsa-MIR574 or pLV-miR-574-5p-shRNA treated versus pLV-miR-shRNA-ctrl treated (n ＝ 3-4) . FIG. 3b shows the verification of lentivirus-mediated knockdown of miR-574-5p in HeLa cells prepared for microarray analyses. HeLa cells were seeded and infected with LV-miR-574-5p-shRNA or control virus. 96 hours after the infection, cells were harvested for analyses. **, p < 0.01； LV-miR-574-5p-shRNA treated versus LV-miR-shRNA-ctrl treated (n ＝ 3) .

FIG. 4 illustrates the stimulation of immune response by miR-574p-5p as determined by analyses. FIG. 4a is a figure of western blots showing plasmid-mediated miR-574-5p overexpression stimulated tyrosin-701 phosphorylated STAT1 and total STAT1 expression whereas miR-574-5p knockdown suppressed the expression of MyD88, TRAF3, tyrosin-701 phosphorylated STAT1 and total STAT1 in THP1 monocytic cells, n ＝ 3. FIG. 4b is a figure of statistical analyses corresponding to the western blots described above. NS, not significant； *, p < 0.05； **, p < 0.01； ***, p < 0.001, pFlag-CMV2-transfected versus phsa-MIR574-transfected or LV-miR-shRNA-ctrl transfected versus LV-miR-574-5p-shRNA-transfected.

FIG. 5 illustrates the luciferase reporter assays of miR-574p-5p in regulating immune and inflammatory responses through activating hTLR8. FIG. 5a shows that plasmid-mediated miR-574-5p overexpression stimulated whereas miR-574-5p knockdown suppressed NFκB and interferon-mediated transcriptional activities in human THP1 monocytic cells. THP1 cells were co-transfected with an indicated plasmid together with a luciferase reporter plasmid and pSV40-β-galactosidase (4: 3: 1) and incubated for 24 h. Cells were harvested for the luciferase reporter assays and β-galactosidase activity assay. NS, not significant； *, p < 0.05； **, p < 0.01； ***, p < 0.001, pFlag-CMV2 versus phsa-MIR574 or pLV-miR-shRNA-ctrl versus pLV-miR-574-5p-shRNA, n ＝ 3. FIG. 5b shows that plasmid-mediated miR-574-5p overexpression stimulated NFκB and interferon-mediated transcriptional activities in HEK-Blue-TLR8 cells but not HEK-Blue-TLR7 cells (except that for IFNβ) (n ＝ 4) . THP1 cells were transfected with indicated plasmid for 24 h, or infected with LV-miR-574-5p-shRNA or control lentivirus for 96 h. Cells were harvested for western blotting assays. NS, not significant； *, p < 0.05； **, p < 0.01； ***, p < 0.001, pFlag-CMV2-transfected versus phsa-MIR574-transfected. FIG. 5c shows that Dotap-conjugated miR-574-5p transfection stimulated NFκB transcriptional activity in HEK-Blue-TLR8 cells but not HEK-Blue-TLR7 cells (n ＝ 3) . HEK-Blue-TLR7 or HEK-Blue-TLR8 cells were grown and co-transfected with a luciferase reporter plasmid (pNFκB-luc) and pSV40-β-galactosidase (3: 1) . 24 h after the transfection, cells were stimulated with 1 μg/ml of R848 or 10 μg/ml of Dotap-PS-miR-16 or Dotap-PS-miR-574-5p and in the absence or presence of 4 mM of uridine for 8 h. MiR-16 served as a negative control whereas R848 served as a positive control. Cells were collected for luciferase activity assay. NS, not significant； *, p < 0.05； **, p < 0.01； ***, p < 0.001, compared with an appropriate Control medium group； ###, p < 0.001, Dotap-PS-miR-574-5p versus Dotap-PS-miR-574-5p + uridine.

FIG. 6 illustrates the effects of knocking-down hTLR8 or hTLR7 on NFκB-mediated transcriptional activities as determined by the luciferase reporter assays. FIG. 6a shows effects of hTLR7 knocking-down by shRNA constructs in HEK-Blue-TLR7 cells. Cells were transfected as indicated. 24 hours after the transfection, cells were harvested for qPCR analyses. Statistical comparisons were made for pLV-sh-ctrl versus pLV-sh-hTLR7-1/2. ***, p < 0.001； n ＝ 3-4. FIG. 6b shows effects of hTLR8 knocking-down by shRNA constructs in HEK-Blue-TLR8 cells (n ＝ 3-4) . Cells were transfected as indicated. 24 hours after the transfection, cells were harvested for qPCR analyses. Statistical comparisons were made between pLV-sh-ctrl versus pLV-sh-hTLR8-1/2/3. Statistical comparisons were made for pLV-sh-ctrl versus pLV-sh-hTLR7-1/2. ***, p < 0.001； n ＝ 3-4. FIG. 6c shows effects of hTLR7 knocking-down by shRNA constructs in HEK-Blue-TLR7 cells. Cells were transfected as indicated. 24 hours after the transfection, cells were harvested for qPCR analyses. NS, not significant； ***, p < 0.001； compared to pLV-sh-ctrl +Dotap-PS-miR-574-5p or pLV-sh-ctrl + R848, n ＝ 3-4. FIG. 6d shows effects of hTLR8 knocking-down by shRNA constructs in HEK-Blue-TLR8 cells. Cells were transfected as indicated. 24 hours after the transfection, cells were harvested for qPCR analyses. NS, not significant； ***, p < 0.001； compared to pLV-sh-ctrl + Dotap-PS-miR-574-5p or pLV-sh-ctrl + R848, n ＝ 3-4.

FIG. 7 illustrates ELISA assay of effects of miR-574-5p on the secretion of IFNα/γ, TNFαand IL/6 in hPBMCs. hPBMCs were treated with 1 μg/ml R848 or 10 μg/ml of Dotap-PS-miR-16 or Dotap-PS-miR-574-5p for 24 h. Alternatively, hPBMCs were infected with LV-miR-ctrl or LV-MIR574 for 96 h. Subsequently, the media were harvested for ELISA analyses. NS, not significant； *, p < 0.05； **, p < 0.01； ***, p < 0.001； compared with control medium-treated or Dotap only-treated or LV-miR-shRNA-ctrl versus LV-miR-574-5p-shRNA, n ＝ 3. FIG. 7a: IFNα. FIG. 7b: IFNγ. FIG. 7c: TNFα. FIG. 7d: IL6.

FIG. 8 illustrates flow cytometry analyses of the effects of miR-574-5p on TNFα-secreting hPBMCs. About 1×10⁶ hPBMCs were seeded in 6-well plate and treated with 1 μg/ml R848 or 10 μg/ml of Dotap-PS-miR-574-5p or Dotap-only. 24 h after the treatment, cells were harvested for flow cytometry analyses. FIG. 8a is typical flow cytometry result. FIG. 8b Statistical analyses of the flow cytometry analysis showing that miR-574-5p exposure significantly increased the percentage of TNFα-secreting hPBMCs. NS, not significant； *, p < 0.05； **, p < 0.01； ***, p < 0.001； compared to Dotap-only, n ＝ 3.

FIG. 9 illustrates flow cytometry analyses of effects of miR-574-5p on the alterations in the distribution of immune cells in hPBMCs. FIG. 9a shows altered immune cell distribution in hPMBCs following stimulation with miR-574-5p. About 1×10⁶ hPBMCs were seeded in 6-well plate and treated with 1 μg/ml R848 or 10 μg/ml of Dotap-PS-miR-16 or Dotap-PS-miR-574-5p. 24 h after the treatment, cells were harvested for flow cytometry analyses. T-helper cells, T_h (CD3⁺CD4⁺) ； cytotoxic T-cells, T_c (CD3⁺CD8⁺) ； natural killer cells, NK, (CD3^-CD56⁺) ； natural killer T-cells, NKT (CD3⁺CD56⁺) and regulatory T-cells, T_reg (CD4⁺CD25⁺) . NS, not significant； *, p < 0.05； **, p < 0.01； ***, p < 0.001； compared to Dotap-PS-miR-16 or Dotap only, n ＝ 3. FIG. 9b is a figure of representative flow cytometry showing miR-574-5p induced alterations in the distribution of immune cells in hPMBCs.

FIG. 10 illustrates ELISA assays showing miR-574p-5p stimulated cytokine secretion in mouse macrophages and mPBMCs. FIG. 10a shows miR-574-5p exposure significantly induced TNFα and IL6 secretion in mouse peritoneal macrophages (n ＝ 3-4) . About 1×10⁵ mouse peritoneal macrophages were seeded and treated with 10 μg/ml of Dotap-PS-miR-574-5p or 1 μg/ml of R848 in 96-well plates. 24 h after the stimulation, the media was collected for ELISA analyses. **, p < 0.01； compared with B6. WT + Dotap. FIG. 10b shows Tnfα secretion in cultured RAW264.7 macrophages stimulated with 1 μg/ml R848 and 10 μg/ml Dotap-PS-miR-574-5p + 4 mM uridine for 24 h. ***, p < 0.01； compared with Dotap + uridine treated, n ＝ 3-4. (c) shows Tnfα and IL6 secretion in mPBMCs stimulated with 10 μg/ml Dotap-miR-16 or Doatp-PS-miR-574-5p for 24 h. ***, p < 0.01； compared with Dotap-only, n ＝ 3-6.

FIG. 11 illustrates the activation of mTLR7 by miR-574-5p to regulate immune and inflammatory responses in mice. FIG. 11a shows that lentivirus-mediated overexpression of miR-574-5p greatly increased serum levels of TNFα and IL6 in wild-type C57BL/6 mice but in mTLR7 knockout mice the stimulation was greatly attenuated. NS, not significant； ***, p < 0.001, compared to B6. WT + LV-MIR-ctrl or B6. mTLR7^-/-+ LV-MIR-ctrl； ###, p < 0.001, compared to B6. WT + LV-hsa-MIR574； n ＝ 4-6. FIG. 11b &c show that miR-574-5p exposure significantly increased TNFα-secreting mBMDCs (flow cytometry) and TNFα secretion (ELISA) by mBMDCs in the B6. WT mice but not the B6. mTLR7^-/-mice (n ＝ 3) . About 1×10⁵ or 1×10⁶ mBMDCs were seeded in 6-well plate and treated with 10 μg/ml of Dotap-PS-miR-574-5p or 100 ng/ml of LPS. 24 h after the treatment, cells were harvested for flow cytometry or ELISA analyses. NS, not significant； **, p < 0.01； ***, p < 0.001, compared with either B6. WT + Dotap or B6. mTLR7^-/- + Dotap. ##, p <0.01； ###, p < 0.001； B6. WT + Dotap-PS-miR-574-5p versus B6. mTLR7^-/- +Dotap-PS-miR-574-5p. FIG. 11d shows that miR-574-5p exposure significantly increased CD69 positive splenocytes in the B6. WT mice but not the B6. mTLR7^-/-mice (n ＝ 4) . About 1×10⁶ mouse splenocytes were seeded in 6-well plate and treated with 10 μg/ml of Dotap-PS-miR-574-5p or miR-16.24 h after the treatment, cells were harvested for flow cytometry analyses. *, p < 0.05； ***, p < 0.001, compared with either B6. WT + Dotap or B6.mTLR7^-/- + Dotap. ###, p < 0.001； B6. WT + Dotap-PS-miR-574-5p versus B6.mTLR7^-/- + Dotap-PS-miR-574-5p.

FIG. 12 illustrates representative flow cytometry analyses showing the effects of miR-574-5p on stimulating bone marrow-derived dendritic cells (BMDC) and splenic T lymphocytes. FIG. 12a shows that miR-574-5p exposure significantly stimulated TNFαsecretion in CD11c⁺ BMDCs from the wild-type mice but in mTLR7 knockout mice the effects were greatly reduced. FIG. 12b shows that miR-574-5p exposure significantly stimulated the activation (CD69) of splenic T lymphocyte from the wild-type mice but in mTLR7 knockout mice the effects were greatly reduced.

FIG. 13 illustrates analyses of mRNA or protein expression in miR-574-5p-knocked-down HeLa cells by microarray hybridization, qPCR or Western blots. CCL2, (C-C Motif) ligand-2； CD74, cluster of differentiation-74； HLA-DRA, HLA class II histocompatibility antigen, DR alpha chain； HLA-C, major histocompatibility complex, Class I, C； IL8, interleukin-8； IRF8, interferon regulatory factor-8； NR4A1, nuclear receptor subfamily-4, group-A, member-1； OLR1, oxidized low-density lipoprotein receptor-1； SFRS1, serine/arginine-rich splicing factor-1； TSC1, tuberous sclerosis-1. NS, not significant； *, p < 0.05； **, p < 0.01； ***, p < 0.001, LV-miR-shRNA-ctrl versus LV-miR-574-5p-shRNA. FIG. 13a is a volcano plot of mRNA expression in miR-574-5p knockdown HeLa cells as determined by NimbleGen 12x135K microarray hybridization (n ＝ 3) . Array hybridization and data processing were performed according to routine methods well known by those skilled in the art. The red point in the plot represents the differentially expressed genes with statistical significance. The vertical green lines in the plot divided genes that were up-regulated (151 genes) or down-regulated (661 genes) by at least 2 folds, respectively, whereas the horizontal green line represents a p-value of 0.05. FIG. 13b shows western blots analyses of protein expression of selected genes in miR-574-5p knockdown HeLa cells. FIG. 13c shows qPCR analyses of mRNA expression of selected genes in miR-574-5p knockdown HeLa cells (n ＝ 3-5) . FIG. 13d shows enrichment of the 661 significantly down-regulated genes in miR-574-5p knockdown HeLa cells in 30 signaling or disease pathways.

FIG. 14 illustrates the relevancy between aberrant miR-574-5p signaling and cervical cancer development. FIG. 14a shows miR-574-5p expression in cervical cancer tissues and their adjacent normal tissues from 18 human patients as determined by qPCR. Data represented mean + SEM for three replicates. *, p < 0.05； **, p < 0.01； ***, p < 0.001, adjacent normal versus cervical tumor. FIG. 14b shows that knocking-down of miR-574-5p greatly reduced tumor growth in the nude mice inoculated with HeLa cells. Athymic BALB/c nude mice were obtained from the SLAC Laboratory Animals Co Ltd, Shanghai, China. 8-wk old male nude mice were subcutaneously inoculated with 2×10⁶ HeLa cells stably transduced with either LV-miR-shRNA-ctrl (left side, up-arrow indicated) or LV-miR-574-5p-shRNA (right side, down-arrow indicated) onto the dorsal flanks of animals. Tumors were dissected 4 wk after the inoculation.

FIG. 15 illustrates qPCR analyses of the levels of miR-574-5p in the serum samples from human SLE patients and the serum and other tissues of female lupus-prone B6. Fas^lpr/^lpr mice. FIG. 15a shows serum levels of miR-574-5p in normal healthy individuals and SLE patients as determined by qPCR. **, p < 0.01, normal healthy individuals versus SLE patients, n ＝ 11. FIG. 15b shows serum levels of miR-574-5p in the B6. WT or B6. Fas^lpr/lpr mice at ages of 90-d and 180-d as determined by qPCR. *, p < 0.05, ***, p < 0.001, B6. WT versus B6. Fas^lpr/lpr； n ＝ 3-6. FIG. 15c shows kidney levels of miR-574-5p in the B6. WT or B6.Fas^lpr/lpr mice at ages of 90-d and 180-d as determined by qPCR. *, p < 0.05, ***, p <0.001, B6. WT versus B6. Fas^lpr/lpr； n ＝ 3-6. FIG. 15d shows miR-574-5p levels in the brain, heart, liver, lung, lymph node and spleen tissues of the B6. WT or B6. Fas^lpr/lpr mice at the age of 90-d. NS, not significant； *, p < 0.05； B6. WT versus B6. Fas^lpr/lpr； n ＝ 6-7. FIG. 15e shows miR-574-5p levels in the brain, heart, liver, lung, lymph node and spleen tissues of the B6. WT or B6. Fas^lpr/lprmice at the age of 180-d. NS, not significant； *, p < 0.05, **, p <0.01, B6. WT versus B6. Fas^lpr/lpr； n ＝ 6-7.

FIG. 16 illustrates the effects of knockdown of miR-574-5p on ameliorating SLE and lupus nephritis associated parameters in the B6. Fas^lpr/^lpr mice at the age of 20-wk. NS, not significant； **, p < 0.01； ***, p < 0.001, LV-miR-shRNA-ctrl versus LV-miR-574-5p-shRNA. In vivo silencing of miR-574-5p was achieved by treatment with lentiviruses carrying shRNA against miR-574-5p as described. FIG. 16a shows lentivirus-mediated knockdown of miR-574-5p in the kidney and liver of B6. Fas^lpr/^lpr mice (n ＝ 6) . FIG. 16b shows that inhibition of miR-574-5p significantly ameliorated lupus-associated splenomegaly (n ＝ 6) . FIG. 16c-h show that silencing of miR-574-5p in the lupus-prone B6. Fas^lpr/lpr mice led to reduced serum anti-dsDNA autoantibody, blood urea nitrogen, proteinuria, serum TNFα, IL6 and IFNα (n ＝ 5-7) , although the differences for proteinuria and serum IFNα were not statistically significant.

FIG. 17 illustrates histochemical and immunohistochemical staining analyses of the renal tissues and liver tissues in untreated B6. WT, LV-miR-shRNA-ctrl-treated B6. Fas^lpr/lpr and LV-miR-574-5p-shRNA treated B6. Fas^lpr/lpr at the age of 20-wk. Results were typical for at least three mice. FIG. 17a shows histochemical staining of the renal cortex by the PAS staining and immunohistochemistry staining of the renal cortex by anti-IgG antibody. FIG. 17b shows histochemical staining of the renal medulla by the PAS staining and immunohistochemistry staining of the renal medulla by anti-IgG antibody. FIG. 17c shows immunohistochemical staining of renal cortex by anti-CD68 antibody. FIG. 17d shows immunohistochemical staining of the liver tissues by anti-CD68 antibody.

FIG. 18 illustrates flow cytometry analyses of distribution of splenic immune cells in the female B6. Fas^lpr/lpr mice as a consequence of miR-574-5p knockdown. FIG. 18a shows alterations in splenic immune cells in LV-miR-shRNA-ctrl-treated B6. Fas^lpr/lpr and LV-miR-574-5p-shRNA treated B6. Fas^lpr/lpr at the age of 20-wk. Mouse spleen cell suspensions were prepared from the spleen tissues dissected from LV-miR-shRNA-ctrl-treated and LV-miR-574-5p-shRNA treated B6. Fas^lpr/lpr. After the elimination of red blood cells, spleen cells were stained with specific antibodies as indicated and used for flow cytometry analyses. T-helper cells, T_h (CD3⁺CD4⁺) ； cytotoxic T-cells, T_c (CD3⁺CD8⁺) ； natural killer cells, NK, (CD3^-NK1.1⁺) ； natural killer T-cells, NKT (CD3⁺NK1.1⁺) and regulatory T-cells, T_reg (CD4⁺CD25⁺) . NS, not significant； *, p < 0.05； LV-miR-shRNA-ctrl versus LV-miR-574-5p-shRNA treated； n ＝ 6. FIG. 18b shows representative results for flow cytometry analyses of T_h and NK/NKT cells. FIG. 18c shows representative results for flow cytometry analyses of T_c and T_reg cells.

DETAILED DESCRIPTION OF THE INVENTION

The following detailed description and examples are given merely for easy understanding the present invention and illustrating the advantages thereof, not intended to limit the present invention.

Throughout this disclosure, various publications, patents and published patent specifications are referenced by citation. The disclosures of these publications, patents and published patent specifications are hereby incorporated by references.

The inventors found that miR-574-5p, miR-574-5p derivatives, and miR-574-5p inhibitors are effective immunomodulatory compounds. Identification of the immunomodulatory potential of miR-574-5p, miR-574-5p derivatives, and miR-574-5p arose through a series of experiments with miRNAs and mammalian TLR7 and/or mammalian TLR8. As a result of this effort, it has now been discovered that miR-574-5p, miR-574-5p derivatives, and miR-574-5p inhibitors are immune modulatory and act through mammalian TLR7 and/or mammalian TLR8 signaling. Specifically, the invention provides natural or synthetic miR-574-5p as well as miR-574-5p derivatives, and miR-574-5p inhibitors with improved in vivo stability that modulates the immune response through mammalian TLR7 and/or mammalian TLR8. MiR-574-5p and its derivative, when working as agonists of mammalian TLR7 and/or mammalian TLR8, could initiate diverse innate and acquired immune response mechanisms through the activation of a number of immunocytes, with the resultant cytokine and interferon secretion leading to the elimination of the pathogen or tumor cells coupled with the development of antigen-specific antibodies and cell-mediated response. The inhibitors of miR-574-5p (antagomiR-574-5p) , when working as the antagonists of mammalian TLR7 and/or mammalian TLR8 could inhibit unwanted immune and inflammatory response through blocking aberrant mammalian TLR7 and/or mammalian TLR8 signaling.

The present invention provides compositions and methods for enhancing or attenuating the immune and inflammatory response elicited by miR-574-5p, miR-574-5p derivatives, or miR-574-5p inhibitors used for immunotherapeutics and related applications as in the treatment of cancer, autoimmune disorders, asthma, allergies, and bacteria, parasitic, and viral infections. The miR-574-5p, miR-574-5p derivatives can be used as adjuvants in combination with other agents useful for the treatment and prevention of diseases or conditions that involve immune response modulation, especially via mammalian TLR7 and/or mammalian TLR8 signaling.

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Any methods, devices and materials similar or equivalent to those described herein can be used in the practice of this invention.

As used herein, the terms “Toll-like receptor” and, equivalently, “TLR” refer to any member of a family of at least ten highly conserved mammalian pattern recognition receptor proteins (TLR1-TLR10) which recognize pathogen-associated molecular patterns (PAMPs) and act as key signaling elements in innate immunity. TLR polypeptides share a characteristic structure that includes an extracellular (extracytoplasmic) domain that has leucine-rich repeats, a transmembrane domain, and an intracellular (cytoplasmic) domain that is involved in TLR signaling. TLRs include but are not limited to human TLRs.

As used herein, the term “agonist” refers to a compound that, in combination with a receptor (e.g., a TLR) , can produce a cellular response. An agonist may be a ligand that directly binds to the receptor. Alternatively, an agonist may combine with a receptor indirectly by, for example, (a) forming a complex with another molecule that directly binds to the receptor, or (b) otherwise resulting in the modification of another compound so that the other compound directly binds to the receptor. An agonist may be referred to as an agonist of a particular TLR (e.g., a TLR7 and/or TLR8 agonist) . In certain embodiments of the invention, the agonists for mTLR7 or hTLR8 can be miR-574-5p or miR-574-5p derivatives.

As used herein, the term “antagonist” refers to a compound that can combine with a receptor to reduce or inhibit a cellular activity. An antagonist may be a ligand that directly binds to the receptor. Alternatively, an antagonist may combine with a receptor indirectly by, for example, (a) forming a complex with another molecule that directly binds to the receptor, or (b) otherwise results in the modification of another compound so that the other compound directly binds to the receptor. In certain embodiments of the invention, the antagonists for mTLR7 or hTLR8 can be miR-574-5p inhibitors.

As used herein, the terms “miR” , “mir” and “miRNA” are used to refer to microRNA, a class of small RNA molecules that are capable of modulating RNA translation.

“miR-574-5p” refers to the mature miRNA having the following nucleic acid sequence:

5’ -UGAGUGUGUGUGUGUGAGUGUGU-3’ .

As used herein, the term “miR-574-5p inhibitors” refers to nucleotides or derivatives thereof that have the ability of silencing miR-574-5p, which can be shRNA against miR-574-5p, single-stranded RNA complementary to miR-574-5p, double-stranded siRNA targeting miR-574-5p, single-stranded DNA complementary to miR-574-5p； the derivatives of the single-stranded RNA complementary to miR-574-5p, double-stranded siRNA targeting miR-574-5p, and single-stranded DNA complementary to miR-574-5p in the forms of phosphorothiolate modification, morpholino modification, 2’ -O-methyl-modification, 2’ -O-methoxyethyl-modification, 2’ -fluoro-modification, LNA-modification； or combinations thereof.

“PS” means phosphorothioate esters linkages through which nucleotides are joined for replacement of phosphodiester linkages. For example, “PS-miR-574-5p” refers to the miRNA having the following nucleobase sequence: 5’ -UsGsAsGsUsGsUsGsUsGsUsGsUs GsUsGsAsGsUsGsUsGsU-3’ , in which “s” represents the phosphorothioate linkages.

“Dotap” means a lipid carrier used as a transfection reagent, whose chemical name is 1, 2-dioleoyl-3-trimethylammoniumpropane.

“Morpholino” means that the RNA sequence is modified with a morpholino group.

“2’ -O-methyl” means that the RNA sequence is modified with an O-methyl group at the 2’ position.

“2’ -O-methoxyethyl” means that the RNA sequence is modified with an O-methoxyethyl group at the 2’ position.

“2’ -fluoro” means that the RNA sequence is modified with a fluoro group at the 2’ position.

As used herein, the term “miR-574-5p-based compound (s) ” refers to the miR-574-5p, miR-574-5p derivatives and miR-574-5p inhibitors disclosed throughout the specification of the present invention.

As used herein, the term “LNA” refers to is a modified RNA nucleotide. The ribose moiety of an LNA nucleotide is modified with an extra bridge connecting the 2’ oxygen and 4’ carbon. The bridge “locks” the ribose in the 3' -endo (North) conformation, which is often found in the A-form duplexes. LNA nucleotides can be mixed with DNA or RNA residues in the oligonucleotide whenever desired and hybridize with DNA or RNA according to Watson-Crick base-pairing rules.

As used herein, the term “adjuvant” generally refers to a substance which, when added to an immunogenic agent such as vaccine, antibacterial agent or antigen enhances or potentiates an immune response to the agent in the recipient host upon exposure to the mixture. TLR agonists are by now an established class of molecules with potential vaccine adjuvant properties. In some embodiments of the invention, miR-574-5p, miR-574-5p derivatives or combination thereof can act as adjuvant.

As used herein, the term “innate immune response” refers to any type of immune response to certain pathogen-associated molecular patterns (PAMPs) . Innate immunity, which is also known in the art as natural or native immunity, involves principally neutrophils, granulocytes, mononuclear phagocytes, dendritic cells, NKT cells, and NK cells. Innate immune responses can include, without limitation, type I interferon production (e.g., IFNα) , neutrophil activation, macrophage activation, phagocytosis, opsonization, complement activation, and any combination thereof.

As used herein, the term “adaptive immune response” refers to any type of antigen-specific immune response. Adaptive immune responses, which are also known in the art as specific immune responses, involve lymphocytes are also characterized by immunological memory, whereby response to a second or subsequent exposure to antigen is more vigorous than the response to a first exposure to the antigen. The term adaptive immune response encompasses both humoral (antibody) immunity and cell-mediated (cellular) immunity.

As used herein, the term “treat” as used in reference to a disorder, disease, or condition means to intervene in such disorder, disease, or condition so as to prevent or slow the development of, to prevent, slow or halt the progression of, or to eliminate the disorder, disease, or condition.

As used herein, the terms “autoimmune disease” and, equivalently, “autoimmune disorder” and “autoimmunity” , refer to immunologically mediated acute or chronic injury to a tissue or organ derived from the host. The terms encompass both cellular and antibody-mediated autoimmune phenomena, as well as organ-specific and organ-nonspecific autoimmunity. Autoimmune diseases include insulin-dependent diabetes mellitus, rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, atherosclerosis, and inflammatory bowel disease. Autoimmune diseases also include, without limitation, ankylosing spondylitis, autoimmune hemolytic anemia, Behcet’s syndrome, Goodpasture's syndrome, Graves’ disease, Guillain-Barre syndrome, Hashimoto's thyroiditis, idiopathic thrombocytopenia, myasthenia gravis, pernicious anemia, polyarteritis nodosa, polymyositis/dermatomyositis, primary biliary sclerosis, psoriasis, sarcoidosis, sclerosing cholangitis, Sjogren’s syndrome, systemic sclerosis (scleroderma and CREST syndrome) , Takayasu’s arteritis, temporal arteritis, and Wegener’s granulomatosis. Autoimmune diseases also include certain immune complex-associated diseases.

As used herein, the term “cancer” refers to a condition in which abnormally replicating cells of host origin are present in a detectable amount in a subject. The cancer can be a malignant or non-malignant cancer. Cancers or tumors include but are not limited to biliary tract cancer； brain cancer； breast cancer； cervical cancer； choriocarcinoma； colon cancer； endometrial cancer； esophageal cancer； gastric (stomach) cancer； intraepithelial neoplasms； leukemias； lymphomas； liver cancer； lung cancer (e.g., small cell and non-small cell) ； melanoma； neuroblastomas； oral cancer； ovarian cancer； pancreatic cancer； prostate cancer； rectal cancer； renal (kidney) cancer； sarcomas； skin cancer； testicular cancer； thyroid cancer； as well as other carcinomas and sarcomas. Cancers can be primary or metastatic.

As used herein, the terms “infection” and, equivalently, “infectious disease” refer to a condition in which an infectious organism or agent is present in a detectable amount in the blood or in a normally sterile tissue or normally sterile compartment of a subject. Infectious organisms and agents include viruses, bacteria, fungi, and parasites. The terms encompass both acute and chronic infections, as well as sepsis.

As used herein, the term “cytokine” refers to any of a number of soluble proteins or glycoproteins that act on immune cells through specific receptors to affect the state of activation and function of the immune cells. Cytokines include interferons, interleukins, tumor necrosis factor, transforming growth factor beta, colony-stimulating factors (CSFs) , chemokines, as well as others. Various cytokines affect innate immunity, acquired immunity, or both. Cytokines specifically include, without limitation, IFNα, IFNβ, IFNγ, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12, IL-13, IL-18, TNFα, TGFβ, granulocyte colony-stimulating factor (G-CSF) , and granulocyte-macrophage colony-stimulating factor (GM-CSF) .

“hPBMCs” means human peripheral blood mononuclear cells, and “mPBMCs” means mouse peripheral blood mononuclear cells.

As used herein, “effective amount” refers to any amount that is necessary or sufficient for achieving or promoting a desired outcome. In some instances an effective amount is a therapeutically effective amount. A therapeutically effective amount is any amount that is necessary or sufficient for promoting or achieving a desired biological response in a subject. The effective amount for any particular application can vary depending on such factors as the disease or condition being treated, the particular agent being administered, the size of the subject, or the severity of the disease or condition. One of ordinary skill in the art can empirically determine the effective amount of a particular agent without necessitating undue experimentation.

As used herein, the term “subject” refers to a vertebrate animal. In one embodiment the subject is a mammal. In one embodiment the subject is a human. In other embodiments the subject is a non-human vertebrate animal, including, without limitation, non-human primates, laboratory animals, livestock, domesticated animals, and non-domesticated animals.

As used herein, the term “TLR7 and/or TLR8 ligand” , “ligand for TLR7 and/or TLR8” or “TLR7 and/or TLR8 agonist or antagonist” refers to a molecule that interacts directly or indirectly with TLR7 and/or TLR8 through a TLR7 and/or TLR8 domain, and induces TLR7-and/or TLR8-mediated signaling. In certain embodiments, a TLR7 and/or TLR8 ligand is a natural ligand, i.e., a TLR7 and/or TLR8 ligand that is found in nature. In certain embodiments, a TLR7 and/or TLR8 ligand refers to a molecule other than a natural ligand of TLR7 and/or TLR8, e.g., a molecule prepared by human activity.

The term “transfection” refers to the uptake of DNA or RNA by a cell. A cell has been “transfected” when exogenous (i.e., foreign) DNA or RNA has been introduced inside the cell membrane. Transfection can be either transient (i.e., the introduced DNA or RNA remains extrachromosomal and is diluted out during cell division) or stable (i.e., the introduced DNA or RNA integrates into the cell genome or is maintained as a stable episomal element) .

The term “miR-574-5p inhibitor” refers to anti-miR-574-5p or antagomiR-574-5p, which include but not limit to single-stranded RNA complementary to miR-574-5p, single stranded DNA complementary to miR-574-5p, short-hairpin RNA against miR-574-5p, double stranded small-interfering RNA against miR-574-5p and the derivatives of single-stranded RNA complementary to miR-574-5p, the derivatives of single stranded DNA complementary to miR-574-5p, the derivatives of short-hairpin RNA against miR-574-5p, the derivatives of double stranded small-interfering RNA against miR-574-5p.

EQUIVALENTS

While the foregoing invention has been described in detail for purposes of clarity and understanding, it will be appreciated by one skilled in the art from a reading of this disclosure that various changes in form and detail can be made without departing from the true scope of the invention and appended claims. The advantages of the invention are not necessarily encompassed by each embodiment of the invention.

All references, patents and patent publications that are cited in this application are incorporated in their entirety herein by reference.

The invention will now be illustrated by the following non-limiting examples.

EXAMPLES

The examples below are intended to further illustrate certain exemplar embodiments of the invention, and are not intended to limit the scope of the invention.

The materials and methods employed in the present invention are described as follows.

Animals and treatments

Unless indicated otherwise, mice were housed in the specific pathogen-free conditions in the Xiamen University Laboratory Animal Center, with a 12 h-12 h light-dark cycle and regular chow and water provided at libitum. All experimental procedures involving animals were performed in accordance with animal protocols approved by the Institutional Animal Use and Care Committee of Xiamen University.

Lupus-prone mice (B6. MRL-Fas^lpr/J or B6. Fas^lpr/lpr) were obtained from Nanjing University, Nanjing, Jiangsu, China whereas mTLR7 deficient mice (B6.129S1-TLR7^tm1Flv/J, B6. mTLR7^-/-) were purchased from the Jackson Lab (Cat#008380, Jackson Lab, Bar Harbor, Maine, USA) . Normal wild-type C57BL/6 mice (B6. WT) were used as controls for both B6. mTLR7^-/-and B6. Fas^lpr/lpr.

For miR-574-5p overexpression in vivo, 10-wk old male B6. mTLR7^-/-or B6. WT mice were infected with lentiviruses overexpressing miR-574-5p or the control viruses at a dosage of 1 ×10⁷ transforming unit (TU) /mouse once by intravenous injection. 72 h after the lentiviral administration, mice were sacrificed and serum and tissue samples were collected for analyses.

To knockdown miR-574-5p in vivo, 8-wk old female B6. Fas^lpr/lpr mice were administered intravenously with 2×10⁶ TU/mouse of LV-miR-574-5p-shRNA or its control lentiviruses LV-miR-shRNA-ctrl once every two weeks up to a total of 1.2×10⁷ TU/mouse. 2 weeks after the final injection, serum, urine, kidney and liver tissue samples were collected from 20-wk old mice for analyses. Urine was collected by bladder message. Blood samples were collected by sinus puncture.

Clinical samples

All clinical samples were collected with the informed consent of the patients and study protocols that were in accordance with the ethical guidelines of the Declaration of Helsinki (1975) and were approved by the Institutional Medical Ethics Committee of the Fujian Medical University. Human patients with SLE and healthy controls without any age or gender grouping were recruited by the Xiehe Hospital of Fujian Medical University at Fuzhou, China. All SLE patients fulfilled the 1997 American College of Rheumatology revised criteria for SLE. Patients with other diseases or concurrent infection were excluded. Blood samples were obtained from all participants after informed consent. For the isolation of hPBMCs, blood samples from multiple healthy human subjects were combined.

Cell culture, transfection and treatment

Mouse macrophage RAW264.7, human monocytic THP1, cervical cancer HeLa and embryonic kidney (HEK293T) cells were purchased from American Type Culture Collection (Manassas, VA, USA) and maintained as instructed. HEK293T cells overexpressing hTLR7 and/or TLR8 (re-designated as HEK-Blue-hTLR7 and/or TLR8) were purchased from InvivoGen (San Diego, California, USA) and cultured in DMEM supplemented with 10％ (vol/vol) FBS, Normocin (50 μg/mL) , Blasticidin (10 μg/mL) , and Zeocin (100 μg/mL) (InvivoGen) .

Human or mouse peripheral blood mononuclear cells (hPBMCs or mPBMCs) were isolated from the whole blood of healthy human donors or mice by centrifugation through a Ficoll-hypaque gradient centrifugation. hPBMCs/mPBMCs were cultured in RPMI1640 (Gibco, Grand Island, NY, USA) supplemented with 10％ (vol/vol) fetal bovine serum (FBS) .

Mouse peritoneal cells were harvested by peritoneal lavage in 7-10 wk old female B6. WT mice with 8–10 mL of ice-cold PBS. Peritoneal macrophages were centrifuged at 350 ×g for 5 min and the resulting peritoneal macrophages were replated at 5 × 10⁵ cells/ml in DMEM supplemented with 10％ FBS, 2 mM L-glutamine and 100 U/ml penicillin/100 μg/ml streptomycin (all purchased from Sangon Biotech, Shanghai, China) and cultured overnight prior to transfection or stimulation.

Cell suspensions were prepared from the spleen tissues dissected from 7-10 wk old female B6.WT and B6. mTLR7^-/-mice. Red blood cells were eliminated by osmotic lysis using red blood cell lysis buffer (Cat#00-4300-54, eBioscience, San Diego, CA, USA) for 5 min. Splenocytes were obtained by centrifugation at 350×g for 5 min and the resulting cells were plated at 5 × 10⁵ cells/ml in DMEM supplemented with 10％ FBS, 2 mM L-glutamine and 100 U/ml penicillin/100 μg/ml streptomycin. Mouse splenocytes were cultured overnight prior to transfection or stimulation.

Mouse bone marrow-derived dendritic cells (mBMDCs) were prepared by methods described previously. Briefly, bone marrow cells obtained from mouse tibias and femurs from 7-10 wk old female B6. WT and B6. mTLR7^-/-mice were passed through a nylon mesh to remove debris, and approximately 3×10⁶cells were placed in 6-well plates containing 3 ml dendritic cell medium (RPMI1640 supplemented with 10％ FBS, 10 ng/ml GM-CSF (Cat#14-8331-62, eBioscience, San Diego, CA, USA) and 10 ng/ml IL4 (Cat#14-8041-62, eBioscience, San Diego, CA, USA) ) . On day-4 and day-7, 50％ of the medium was replaced with fresh media. On day-7 or day-8, the loosely adherent clusters were dislodged and harvested gently for subsequent experiments.

Lipofectamine-3000 reagent (Invitrogen, Carlsbad, CA, USA) was used for plasmid DNA transfections. Phosphorothioated or digoxin labeled miRNAs chemically synthesized by Invitrogen, Guangzhou, Guangdong, China or Genscript, Nanjing, Jiangsu China as listed in Table 1, according to the manufacturer’s protocols. Cationic lipid N- [1- (2, 3-Dioleoyloxy) propyl] -N, N, N-trimethylammoniummethyl-sulfate (Dotap, Liposomal Transfection Reagent, Cat#1202375, Roche, Nonnenwald, Penzberg, Germany) was used for the conjugation of phosphorothioated miRNAs. Unless otherwise indicated, Dotap-conjugated and phosphorothioated miRNAs were used for cell transfection at the concentration of 10 μg/ml.

TLR7 and/or TLR8 dual-agonist resiquimod (R848, Cat#tlrl-r848, Invivogen, San Diego, CA, USA) was used in cell studies at the working concentration of 1 μg/ml. Lipopolysaccharide (LPS) was purchased from Sigma-Aldrich (Cat#L2630, Sigma-Aldrich, St Louis, MO, USA) and used in cell cultures at the concentration of 100 ng/ml.

Construction of plasmids and lentiviruses

Full-length human hTLR7 and/or TLR8/9-overexpressing plasmids pFlag-hTLR7, pFlag-hTLR8 and pFlag-hTLR9 were generous gifts of Prof. Jiahuai Han, Xiamen University.

To create a plasmid overexpressing the extracellular domain (not including the transmembrane and the cytosolic domains) of hTLR8, a truncated hTLR8 (amino acids 27-827, hTLR8^27-827) was amplified from pFlag-hTLR8 whereas a protein A (PA) cDNA fragment was amplified from Staphylococcus aureus subsp. Aureus (Cat#USA300_TCH1516, ATCC) using primers listed in Table 3. hTLR8^27-827 and PA cDNA fragments were fused by overlapping PCR and the resultant hTLR8^27-827-PA fragment was inserted into the Drosophila Expression System vector pMT-BIP-V5-His (Cat#V413020, Biofeng, Shanghai, China) of using BglII and EcoRI restriction sites to generate PA-and His-double-tagged hTLR8^27-827 overexpressing plasmid phTLR8^27-827-PA-His. In a similar way, other plasmids containing the truncated hTLR7 or mTLR7 and/or TLR8, which include phTLR7^27-838-PA-His, pmTLR7^27-839-PA-His and pmTLR8^27-818-PA-His, were prepared using primers listed in Table 3.

Plasmids overexpressing human miR-574-5p (phsa-MIR574) , mouse miR-574-5p (pFlag-Mir574) and lentiviruses carrying shRNAs for miR-574-5p (LV-miR-574-5p-shRNA) or a negative control shRNA (LV-miR-shRNA-ctrl) were as described previously. To construct a lentiviral vector overexpressing miR-574-5p, a 345-bp human MIR574 gene DNA fragment was PCR amplified from human genomic DNA with the primers listed in Table 3. The PCR-amplified fragment was inserted to a lentiviral vector pLV-EF1a-MCS-IRES-Puro (pLV-MIR-ctrl) to generate pLV-hsa-MIR574. Viral vector pLV-hsa-MIR574 or pLV-MIR-ctrl as well as three lentivirus packaging plasmids (pMDL, pVSVG and pREV) were co-transfected into HEK293T cells. pLV-EF1a-MCS-IRES-Puro, pMDL, pVSVG and pREV were kind gifts from Prof. Jiahuai Han. Media containing lentiviruses (LV-hsa-MIR574 and LV-MIR-ctrl) were collected every 24 h for 3 times and the lentiviruses were purified by ultra-speed centrifugation.

Plasmid vectors expressing small hairpin RNA (shRNA) against hTLR7 (NM_016562.3) and hTLR8 (NM_016610.3) were constructed by inserting chemically-synthesized double-strand DNA fragments containing hTLR7 and hTLR8-targeting shRNA sequences as listed in Table 2 into plasmid pLentiLox3.7 at the HapI and XhoI sites, generating plasmids pLV-sh-hTLR7-1, pLV-sh-hTLR7-2, pLV-sh-hTLR8-1, pLV-sh-hTLR8-2, pLV-sh-hTLR8-3 and pLV-sh-ctrl. The inserted DNA fragments were verified by DNA sequencing.

mRNA microarray analyses in miR-574-5p-knockeddown HeLa cells

HeLa cells were seeded on plates at the density of 20-30％ confluency and incubated overnight. Lentiviral infection of HeLa cells was performed with lentiviruses LV-miR-574-5p-shRNA or LV-miR-shRNA-ctrl at multiplicity of infection of 1: 1 and in the presence of Polyberene (Cat#107689, Sigma-Aldrich, St Louis, MO, USA) . 96 h after viral infection, cells were harvested and total RNAs were prepared for miR-574-5p and mRNA expression analyses by qPCR and microarray analyses.

Microarray analyses of mRNA expression of miR-574-5p-knockdown HeLa cells were performed with NimbleGen 12x135K microarrays (Roche NimbleGen, Inc., Madison, WI, USA) by the KangChen Biotech (Shanghai, China) . Briefly, total RNA of each sample was used for labeling and array hybridization as the following steps: 1) Reverse transcription with by Invitrogen Superscript ds-cDNA synthesis kit； 2) ds-cDNA labeling with NimbleGen one-color DNA labeling kit； 3) Array hybridization using the NimbleGen Hybridization System and followed by washing with the NimbleGen wash buffer kit； 4) Array scanning using the Axon GenePix 4000B microarray scanner (Molecular Devices Corporation) . Scanned images were then imported into NimbleScan software (version 2.5) for grid alignment and expression data analyses. Expression data were normalized through quantile normalization and the Robust Multichip Average algorithm included in the NimbleScan software. Further data analyses were performed using Agilent GeneSpring GX v11.5.1 software. Significant differentially expressed genes were identified through Volcano Plot filtering. Gene ontology analyses and pathway analyses and were applied to determine the enrichment of these differentially expressed genes in these biological pathways.

Real-time quantitative PCR (qPCR)

For qPCR analyses of mRNA, reverse transcription was performed with TRIzol (Invitrogen) -extracted total RNAs using a ReverTra

Kit as instructed (Cat#FSQ-101, Toyobo, Tokyo, Japan) . qPCR was performed using the SYBR Green Real-Time PCR Master Mix (Cat#QPK-212, Toyobo) and the Step One Plus Real-Time PCR system (Applied Biosystems Inc., Foster City, CA, USA) using appropriate primer pairs as listed in Table 4, according to the manufacturers’ protocols and with 18S rRNA as a control.

Serum total RNAs were extracted using a mirVana miRNA isolation kit (Cat#AM1556, Ambion, Austin, TX, USA) whereas the total RNAs from cultured cells or tissues were extracted using TRIzol, according to the manufacturer’s protocols. Five microliters of total RNA was reverse transcribed using the ReverTra

Kit as instructed (TOYOBO, Shanghai, China) and miRNA-specific stem-loop primers listed in Table 4. qPCR was performed with total RNAs, using universal primer and miRNA-specific reverse LNA-primers as listed in Table 4, withU6 RNA served as an internal control. For human serum miR-574-5p analyses by qPCR, serum samples were prepared from blood collected from 11 human SLE patients and 11 healthy controls.

Luciferase reporter assays

A luciferase reporter for NFκB activity (pNFκB-luc) was a kind gift from Prof. Jiahuai Han, Xiamen University whereas reporters for interferon activity (pISRE-luc, pGL3-IFNα-luc and pGL3-IFNβ-luc) were generous gifts from Prof. Rongtuan Lin, McGill University, Montreal, Canada. Luciferase reporter activities in cells co-transfected with any of the luciferase reporter were determined using a luciferase reporter gene assay system (Cat#, Promega, Madison, WI, USA) as instructed. For all luciferase assays, β-galactosidase activities were determined to calibrate for the transfection efficiency. The calibrated value for a proper control was used to normalize all other values to obtain the normalized relative luciferase units (RLU) .

miRNA-TLR protein co-immunoprecipitation (co-IP) assays

To express and purify a truncated hTLR8 protein (the extracellular domain) , Drosophila melanogaster Schneider 2 (S2) cells (Cat#R690-07, Invitrogen, Carlsbad, CA, USA) were transfected with phTLR8^27-827-PA-His, in the presence of plasmids pCoHygro (Cat#K4130-01 Biofeng, shanghai, China) . 48 h after the transfection, cells were selected with Sf-900 II SFM medium (Cat#10902-096, Gibco, Grand Island, NY, USA) containing 300 μg/mL hygromycin (Cat#10687-010 Invitrogen, Carlsbad, CA, USA) to obtain cells stably-overexpressing hTLR8^27-827-PA-His. Approximately 2.5×10⁸ stable cells were induced with CuSO₄at the concentration of 5 mM for 72 h and 50 ml of the media were collected as described. Protein hTLR8^27-827-PA-His was purified with IgG beads (Cat#17-0969-01, GE Healthcare, Connecticut, CT, USA) and resuspended in 200 μl of NT2 buffer and used for miRNA-protein co-IP. In a similar way, truncated proteins hTLR7^27-838-PA-His, mTLR7^27-839-PA-His and mTLR8^27-818-PA-His were overexpressed and purified.

For in vitro co-IP of miRNA with the truncated PA-and His-double tagged-TLRs, firstly 1 μg of 5’ -Dig-labeled miR-16 or miR-574-5p (Dig-miR-16 and Dig-miR-574-5p, Table 1) were mixed with 100 μl above purified hTLR8^27-827-PA-His and 900 μl of in NET2 buffer to allow potential miR-TLR interactions at 4 ℃ for 3 h. Following this, 1μg of anti-Dig antibody was added to the mixtures and incubated at 4 ℃ overnight to allow anti-Dig- (Dig-miR) interactions. Protein A/G beads (Genscript, Nanjing, Jiangsu, China) were subsequently added to the mixtures and incubated for another 5 h. Beads were washed 6 times with the NT2 buffer. The eluted samples were blotted by anti-His and anti-Dig antibodies (Table 5) . Similar co-IPs were performed with truncated proteins hTLR7^27-838-PA-His, mTLR7^27-839-PA-His and mTLR8^27-818-PA-His.

For co-IP of with the full-length TLRs, 5×10⁶ HEK293T cells were seeded and transfected with approximately 5 μg Flag-tagged hTLR7 and/or TLR8/9 overexpressing plasmids or HA-tagged mT7/8/9 overexpressing plasmids. 24 h after the transfection, cells were harvested and lysed by ultrasonification in 300 μl of the polysome lysis buffer on ice, as previously described (Keene JD et al., 2006) . Lysates were centrifuged at 14,000 ×g for 15 min. 100 μl resultant supernatant were incubated with 1 μg of 5’ -Dig-labbled and phosphorothioated miRNAs in NET2 buffer for 3 h at 4 ℃. Subsequently, anti-Dig antibody was added to the supernatants and the mixtures were incubated at 4 ℃ overnight. Pull-down was achieved by incubating the mixtures with protein A/G beads in a final volume of 1 ml of NET2 buffer for 5 h at 4 ℃. Beads were centrifuged and washed six times with NT2 buffer. The eluted samples were analyzed by immunoblots using anti-Dig, anti-Flag or anti-HA antibodies (Table 5) .

Confocal fluorescence microscopy

HeLa cells were seeded onto cell culture plates and grown to 50％ confluence. Cells were then transfected with Dotap-conjugated and Cy3-labelled miR-574-5p (Genscript, Nanjing, China) and incubated for 24 h. Transfected cells were subsequently washed four times with PBS and incubated for 2 h with LysoTracker blue DND-22 (Cat#L7525, Invitrogen) diluted 1:20,000 in PBS, then examined under a confocal microscope.

Flow cytometry

For cell-surface staining, hPBMCs, mBMDCs, macrophages or mouse splenocytes were seeded at the density of 1×10⁶/well on 6-well plate and treated with 10 μg/ml of Dotap-conjugated miR-574-5p or 10 μg/ml Dotap-conjugated miR-16 or 1 μg/ml of R848 or 100 ng/ml of LPS. 24 h after the stimulation, cells were washed, resuspended in cold FACS buffer (PBS containing 0.1％ sodium azide (Cat#S2002, Sigma-Aldrich, St Louis, MO, USA) and 2％ FBS) , and then incubated for 10 min at 4℃. Subsequently, they were stained with antibodies as listed on Table 5 for 30 min. For intracellular staining, cells were washed with cold FACS buffer and fixed in Fixation buffer (Cat#420801, BioLegend, San Diego, CA, USA) at 4℃ for 1 h. Cells were then washed with Permeabilization buffer (Cat#421002, BioLegend, San Diego, CA, USA) and stained with anti-TNFα antibody in Permeabilization buffer at 4℃ for 4 h. The stained cells were washed with FACS buffer and analyzed by flow cytometry (LSRFortessa, BD, San Jose, CA, USA) .

Western blots or immunoblots

Western blots or immunoblots were performed by standard protocols using antibodies listed on Table 5.

ELISA assays of IFNα/γ, TNFα and IL6 and serum anti-dsDNA autoantibody

The levels of human or mouse IFNα/γ, TNFα and IL6 and serum anti-dsDNA were analyzed for supernatants of cell cultures or serum samples using ELISA kits as listed in Table 6 according to the manufacturer’s instructions.

Biochemical parameters, immunohistochemistry and morphometric analyses

To assess renal function, urinary protein (Cat#C035-2, Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China) concentrations and blood urea nitrogen (BUN, Cat#C013-2) were measured using commercial kits from Jiancheng Bioengineering Institute (Nanjing, Jiangsu, China) following the manufacturer’s instructions.

For histological and immunohistological analyses, mice were sacrificed at the age of 20 weeks and kidney and liver tissues were rapidly dissected and fixed in 10％ buffered formalin (v/v) for 24 h before being embedded in paraffin. Kidney tissue sections at 5 μm thickness were stained with periodic acid-Schiff (PAS, Cat#YM0715LA13, Yuanye BioTechnology, Shanghai, China) and examined under a light microscope. For IgG deposits, kidney sections of were incubated with peroxidase-conjugated anti-mouse IgG. Staining was visualized using the chromogenic substrate 3-3’ diaminobenzidine (Cat#1412232031, Maixin Biotech, Fuzhou, Fujian, China) . In addition, kidney and liver tissue sections were de-paraffinized, rehydrated and subjected to antigen retrieval in citrate buffer solution (pH 6.0) . Sections were then incubated with anti-CD68 antibody (Abcam, Cambridge, London, UK) at 1: 200 dilution overnight at 4 ℃, and then processed with the DAB (Streptavidin-Biotin) Detection Kit (Cat#KIT-0017, Maixin Biotech) as instructed.

Other data acquisition, image processing and statistical analyses

Western blot images were captured by Biosense SC8108 GelDocumentation System with GeneScope V1.73 software (Shanghai BioTech, Shanghai, China) . Gel images were imported into Photoshop for orientation and cropping. Data are the means ±SEM. One-way ANOVA with Bonferonni’s post-test was used for multiple comparisons and the Student’s t test (two-tailed) for pair-wise comparisons.

Table 1. A list of chemically-synthesized and HPLC-purified miRNAs purchased from Invitrogen (Guangzhou, China) or Genscript (Nanjing, China) .

Notes: Dig, digoxin； PS, phosphorothioated； s, phosphorothioate linkage.

Table 2. A list of lentiviral shRNA vectors for miRNAs and mRNAs.

Table 4. A list of the primers used for qPCR analyses of mRNAs and miRNAs. LNA, locked nucleic acid.

Table 5. A list of the antibodies used for immunoblots (IB) , immunoprecipitation (IP) , flow cytometry or immunohistochemistry in current study.

Table 6. A list for the ELISA kits used in current study.

Example 1 Similarity analyses of miR-574-5p sequences

Human MIR574 gene is located on human Chromosome 4p. 14 and is the direct neighbor of hTLR1/6/10. The data are based on Ensembl Release 79 (http: //asia. ensembl. org/index. html？ redirect＝no) . Interestingly, not far away, the 4p. 16-15.2 region is previously found to be associated closely with SLE by linkage analyses. Mouse Mir574 gene is located on mouse Chromosome 5q. C31 and is the direct neighbor of mTLR1/6. The data are based on Ensembl Release 79. Sequence alignment showed that miR-574-5p is highly GU-rich and evolutionarily conserved among mammals, bta, Bos taurus (cow) ； cfa, Canis familiaris (dog) ； efu, Eptesicus fuscus (big brown bat) ； ggo, Gorilla gorilla (gorilla) ； hsa, Homo sapiens (human) ； mmu, Mus musculus (mouse) ； ssc, Sus scrofa (pig) . Human and mouse miR-574-5p sequences are based on miRBase 21 (http: //www. mirbase. org) . Sequences for other miR-574-5p sequences are predicted from the precursor sequences of the corresponding mir574 genes. Similarity between mammalian miR-574-5p miRNA sequences and three virus-derived ssRNA sequences showed that miR-574-5p might be potential ligands for mammalian TLR7 or mammalian TLR8. RNA40 is derived from the U5 region of HIV-1 RNA and ssRNA83 and ssRNA120 are derived from SARS coronavirus genome (see FIG. 1) .

Example 2 miR-574-5p is an endogenous ligand for hTLR8 and mTLR7

To determine whether miR-574-5p can serve as a TLR ligand, the ability of miR-574-5p binding to human or mouse TLRs was analyzed. The extracellular domains of hTLR7 and/or TLR8 or mTLR7 and/or TLR8 were firstly fussed with the protein A (PA) from Staphylococcus aureus and expressed the fused proteins in Drosophila melanogaster S2 cells according to the methods described previously. The PA and histidine (His) -double-tagged truncated TLR proteins were purified and incubated with either digoxin (Dig) -labeled miR-574-5p or miR-16 to allow potential miRNA-TLR interactions. RNA-protein co-immunoprecipitation (co-IP) was performed according to routine methods well known by those skilled in the art. Following pull-down of Dig-labeled miR-574-5p or miR-16 by anti-Dig antibodies, immunoblots by anti-His antibody showed that only the extracellular hTLR8 or mTLR7 but not extracellular hTLR7 or mTLR8 were detected in the anti-Dig immunoprecipitates (see FIG. 2a &2c) . In HEK293T cells expressing the full-length hTLR7 and/or TLR8/9 or mTLR7 and/or TLR8/9, similar co-IP assays showed that only hTLR8 and mTLR7 but not the hTLR7/9 or mTLR8/9 was found in the Dig-miR-574-5p-coimmunoprecipiates (see FIG. 2b &2d) . HeLa cells were transfected with Dotap-conjugated Cy3-miR-574-5p (red) and stained with LysoTracker DND-22 (blue) and visualized under a confocal microscope. Confocal microscopy, on the other hand, demonstrated that exogenously-introduced miR-574-5p mainly localized to the endosomes/lysosomes in HeLa cells (see FIG. 2e) , making it possible for miR-574-5p to physical interact with TLR7 or TLR8. Together, these results clearly established that miR-574-5p is capable of specific binding with endosomal hTLR8 or mTLR7, suggesting that miR-574-5p is a novel endogenous ligand for hTLR7 and/or TLR8 and mTLR7 and/or TLR8.

Example 3 Stimulation of immune response by miR-574-5p

To evaluate the functional consequence of miR-574-5p on immune and inflammatory response, miR-574-5p in human monocytic THP1 cells and cervical cancer HeLa cells was firstly overexpressed or knocked-down. HeLa cells were seeded and infected with LV-miR-574-5p-shRNA or control virus. 96 hours after the infection, cells were harvested for analyses. The results were verified by miRNA qPCR. **, p < 0.01； LV-miR-574-5p-shRNA treated versus LV-miR-shRNA-ctrl treated (n ＝ 3) (see FIG. 3a &3b) . Then the western blot analyses and the corresponding statistical analyses were performed, which showed that overexpression of miR-574-5p markedly increased un-phosphorylated signal transducers and activators of transcription-1 (STAT1) and Y⁷⁰¹-phosphorylated STAT1 whereas inhibition of miR-574-5p significantly suppressed protein expression of myeloid differentiation primary response gene-88 (MyD88) , TNF receptor-associated factor-3 (TRAF3) , un-phosphorylated STAT1 and Y⁷⁰¹-phosphorylated STAT1. NS, not significant； *, p < 0.05； **, p < 0.01； ***, p < 0.001, pFlag-CMV2-transfected versus phsa-MIR574-transfected or LV-miR-shRNA-ctrl transfected versus LV-miR-574-5p-shRNA-transfected (see FIG. 4a &4b) .

Example 4 miR-574p-5p and miR-574-5p derivatives regulated immune and inflammatory responses through activating human hTLR8

THP1 cells were co-transfected with an indicated plasmid pLV-miR-574-5p-shRNA or control plasmid shown in FIG. 5a together with a luciferase reporter plasmid and pSV40-β-galactosidase (4: 3: 1) and incubated for 24 h. Cells were harvested for the luciferase reporter assays and β-galactosidase activity assay. Luciferase reporter assays were performed according to routine methods well known by those skilled in the art. Luciferase reporter assays indicated that plasmid-mediated miR-574-5p overexpression stimulated whereas miR-574-5p knockdown suppressed NFκB and interferon-mediated transcriptional activities in THP1 monocytic cells. NS, not significant； *, p < 0.05； **, p <0.01； ***, p < 0.001, pFlag-CMV2 versus phsa-MIR574 or pLV-miR-shRNA-ctrl versus pLV-miR-574-5p-shRNA, n ＝ 3 (see FIG. 5a) .

HEK-Blue-hTLR7 or HEK-Blue-hTLR8 cells, which stably expresses hTLR7 or hTLR8 respectively, were transfected with indicated plasmids phsa-MIR574, or pFlag-CMV2 as shown in FIG. 5b respectively for 24 h. Cells were harvested for western blotting assays. Plasmid-mediated miR-574-5p overexpression stimulated NFκB (assayed by the pNFκB-luc reporter) and interferon-mediated transcriptional activities (assayed by the pGL3-ISRE-luc, pGL3-IFNα-luc and pGL3-IFNβ-luc respectively) in HEK-Blue-TLR8 cells but not HEK-Blue-TLR7 cells (except that for IFNβ) (n ＝ 4) (see FIG. 5b) . NS, not significant； *, p < 0.05； **, p < 0.01； ***, p < 0.001, pFlag-CMV2-transfected versus phsa-MIR574-transfected.

These results suggest crucial regulatory roles of miR-574-5p in immune and inflammatory responses. To ascertain the specific activation of hTLR8 by miR-574-5p, transfection studies were performed with Dotap-conjugated miR-574-5p or miR-16 in HEK-Blue-hTLR7 or HEK-Blue-hTLR8 cells. The transfected HEK-Blue-TLR7 or HEK-Blue-TLR8 cells were grown and subsequently co-transfected with a luciferase reporter plasmid (pNFκB-luc) and pSV40-β-galactosidase (3: 1) . 24 h after the transfection, cells were stimulated with 1 μg/ml of R848 or 10 μg/ml of Dotap-PS-miR-16 or Dotap-PS-miR-574-5p and in the absence or presence of 4 mM of uridine for 8 h. MiR-16 served as a negative control whereas R848 served as a positive control. Cells were collected for luciferase activity assay. Luciferase reporter assays indicated that Dotap-conjugated miR-574-5p transfection stimulated NFκB transcriptional activity in HEK-Blue-TLR8 cells but not HEK-Blue-TLR7 cells (n ＝ 3) (see FIG. 5c) . Interestingly, the presence of uridine significantly enhanced the stimulatory effects of miR-574-5p on hTLR8, which is consistent with a recent finding from hTLR8 X-ray crystal structural studies. The specificity of binding and activation of hTLR8 by miR-574-5p were further verified by a series of transfections studies. Together these data established that miR-574-5p is a specific and potent agonist for hTLR8.

Example 5 Knocking-down hTLR8 greatly diminished NFκB-mediated transcriptional activities induced by miR-574-5p

To evaluate the influence of hTLR7 and hTLR8 on NFκB-mediated transcriptional activities, hTLR7 were first knocked-down by shRNA constructs in HEK-Blue-TLR7 cells. Cells were then transfected with indicated plasmids pLV-sh-hTLR7-1, pLV-sh-hTLR7-2, or control plasmid shown in FIG. 6a respectively. 24 hours after the transfection, cells were harvested for qPCR analyses. Statistical comparisons were made for pLV-sh-ctrl versus pLV-sh-hTLR7-1 or pLV-sh-hTLR7-2 (see FIG. 6a) . ***, p < 0.001； n ＝ 3-4.

Second, hTLR8 were knocked-down by shRNA constructs in HEK-Blue-TLR8 cells (n ＝3-4) . Cells were then transfected with indicated plasmids pLV-sh-hTLR8-1, pLV-sh-hTLR8-2, pLV-sh-hTLR8-3, or control plasmid shown in FIG. 6b. 24 hours after the transfection, cells were harvested for qPCR analyses. Statistical comparisons were made for pLV-sh-ctrl versus pLV-sh-hTLR7-1 or pLV-sh-hTLR7-2. Statistical comparisons were made between pLV-sh-ctrl versus pLV-sh-hTLR8-1 or pLV-sh-hTLR8-2 or pLV-sh-hTLR8-3. ***, p < 0.001； n ＝ 3-4 (see FIG. 6b) .

Third, hTLR7 were knocked-down by shRNA constructs in HEK-Blue-TLR7 cells. Cells were transfected with indicated plasmids shown in FIG. 6c. 24 hours after the transfection, cells were harvested for qPCR analyses. NS, not significant； ***, p < 0.001； compared to pLV-sh-ctrl + Dotap-PS-miR-574-5p or pLV-sh-ctrl + R848, n ＝ 3-4 (see FIG. 6c) . The results indicated that hTLR7 knockdown did not significantly affect miR-574-5p-induced NFκB-mediated transcriptional activity in HEK-Blue-hTLR7 cells.

Fourth, hTLR8 were knocked-down by shRNA constructs in HEK-Blue-TLR8 cells. Cells were transfected with indicated plasmids shown in FIG. 6d. 24 hours after the transfection, cells were harvested for qPCR analyses. NS, not significant； ***, p < 0.001； compared to pLV-sh-ctrl + Dotap-PS-miR-574-5p or pLV-sh-ctrl + R848, n ＝ 3-4 (see FIG. 6d) . The results indicated that hTLR8 knockdown significantly affected miR-574-5p-induced NFκB-mediated transcriptional activity in HEK-Blue-hTLR8 cells.

Together these data established that knocking-down hTLR8 but not hTLR7 greatly diminished NFκB-mediated transcriptional activities.

Example 6 Exposure of miR-574-5p and miR-574p-5p derivatives in hPBMCs potently stimulated the secretion of IFNα, IFNγ, TNFα and IL/6

Human hPBMCs were treated with 1 μg/ml R848 or 10 μg/ml of Dotap-PS-miR-16 or Dotap-PS-miR-574-5p for 24 h. Alternatively, hPBMCs were infected with LV-miR-ctrl or LV-miR574 for 96 h. Subsequently, the media were harvested for ELISA analyses. The ELISA analyses showed that miR-574-5p exposure potently stimulated the secretion of IFNα/γ, TNFα and IL/6 in hPBMCs. NS, not significant； *, p < 0.05； **, p < 0.01； ***, p <0.001； compared with control medium-treated or Dotap only-treated or LV-miR-shRNA-ctrl versus LV-miR-574-5p-shRNA, n ＝ 3 (see FIG. 7) .

Example 7 Exposure of miR-574-5p and miR-574-5p derivatives significantly increased the percentage of TNFα-secreting hPBMCs

About 1×10⁶ hPBMCs were seeded in 6-well plate and treated with 1 μg/ml R848 or 10 μg/ml of Dotap-PS-miR-574-5p or Dotap-only. 24 h after the treatment, cells were harvested for flow cytometry analyses. The flow cytometry analyses showed that miR-574-5p exposure significantly increased the percentage of TNFα-secreting hPBMCs. NS, not significant； *, p < 0.05； **, p < 0.01； ***, p < 0.001； compared to Dotap-only, n ＝3 (see FIG. 8) .

Example 8 Exposure of miR-574-5p and miR-574p-5p derivatives caused significant alterations in the distribution of immune cells in hPBMCs

MiR-574-5p was used to stimulate the immune cell redistribution in hPMBCs. About 1×10⁶ hPBMCs were seeded in 6-well plate and treated with 1 μg/ml R848 or 10 μg/ml of Dotap-PS-miR-16 or Dotap-PS-miR-574-5p. 24 h after the treatment, cells were harvested for flow cytometry analyses. T-helper cells, T_h (CD3⁺CD4⁺) ； cytotoxic T-cells, T_c (CD3⁺CD8⁺) ； natural killer cells, NK, (CD3^-CD56⁺) ； natural killer T-cells, NKT (CD3⁺CD56⁺) and regulatory T-cells, T_reg (CD4⁺CD25⁺) . NS, not significant； *, p < 0.05； **, p < 0.01； ***, p < 0.001； compared to Dotap-PS-miR-16 or Dotap only, n ＝ 3 (see FIG. 9a) . FIG. 9b shows representative flow cytometry demonstrating miR-574-5pinduced redistribution of immune cells in hPMBCs.

Example 9 miR-574p-5p and miR-574p-5p derivatives stimulated cytokine secretion in mouse macrophages and mouse peripheral blood mononuclear cells (mPBMCs)

MiR-574-5p was used to induce TNFα and IL6 secretion in mouse peritoneal macrophages (n ＝ 3-4) . About 1×10⁵ mouse peritoneal macrophages were seeded and treated with 10 μg/ml of Dotap-PS-miR-574-5p or 1 μg/ml of R848 in 96-well plates. 24 h after the stimulation, the media was collected for ELISA analyses. **, p < 0.01； compared with Dotap-PS-miR-16 or Dotap-only (see FIG. 10a) .

Tnfα secretion in cultured RAW264.7 macrophages (reportedly express mTLR8 but not mTLR7) were stimulated with 1 μg/ml R848 and 10 μg/ml Doatp-PS-miR-574-5p + 4 mM uridine for 24 h. ***, p < 0.01； compared with Dotap + uridine treated, n ＝ 3-4 (see FIG. 10b) .

Tnfα and IL6 secretion in mPBMCs were stimulated with 10 μg/ml Dotap-miR-16 or Doatp-PS-miR-574-5p for 24 h. ***, p < 0.01； compared with Dotap-only, n ＝ 3-6 (see FIG. 10c) . These ELISA assays showed that miR-574p-5p stimulated cytokine secretion in mouse macrophages and mPBMCs.

Example 10 miR-574p-5p and miR-574p-5p derivatives activated mTLR7 to regulate immune and inflammatory responses in mice

For miR-574-5p overexpression in vivo, 10-wk old male B6. mTLR7^-/-or B6. WT mice were infected with lentiviruses overexpressing miR-574-5p or the control viruses at a dosage of 1 ×10⁷ transforming unit (TU) /mouse once by intravenous injection. 72 h after the lentiviral administration, mice were sacrificed and serum and tissue samples were collected for analyses. Results showed that lentivirus-mediated overexpression of miR-574-5p greatly increased serum levels of TNFα and IL6 in wild-type C57BL/6 mice but in mTLR7 knockout mice the stimulation was greatly attenuated. NS, not significant； ***, p < 0.001, compared to B6. WT + LV-MIR-ctrl or B6. mTLR7^-/-+ LV-MIR-ctrl； ###, p < 0.001, compared to B6. WT + LV-hsa-MIR574； n ＝ 4-6 (see FIG. 11 a) .

About 1×10⁵or 1×10⁶ mBMDCs were seeded in 6-well plate and treated with 10 μg/ml of Dotap-PS-miR-574-5p or 100 ng/ml of LPS. 24 h after the treatment, cells were harvested for flow cytometry or ELISA analyses. Results indicated that miR-574-5p exposure significantly increased TNFα-secreting mBMDCs (flow cytometry) and TNFα secretion (ELISA) by mBMDCs in the B6. WT mice but not the B6. mTLR7^-/-mice (n ＝ 3) . NS, not significant； **, p < 0.01； ***, p < 0.001, compared with either B6. WT + Dotap or B6. mTLR7^-/-+ Dotap. ##, p < 0.01； ###, p < 0.001； B6. WT + Dotap-PS-miR-574-5p versus B6. mTLR7^-/-+ Dotap-PS-miR-574-5p (see FIG. 11 b &c) .

About 1×10⁶ mouse splenocytes were seeded in 6-well plate and treated with 10 μg/ml of Dotap-PS-miR-574-5p or miR-16. 24 h after the treatment, cells were harvested for flow cytometry analyses. Results indicated that miR-574-5p exposure significantly increased CD69 positive splenocytes in the B6. WT mice but not the B6. mTLR7^-/-mice (n ＝ 4) . *, p <0.05； ***, p < 0.001, compared with either B6. WT + Dotap or B6. mTLR7^-/-+ Dotap. ###, p < 0.001； B6. WT + Dotap-PS-miR-574-5p versus B6. mTLR7^-/-+ Dotap-PS-miR-574-5p (see FIG. 11d) .

Example 11 Representative flow cytometry analyses showing stimulation of bone marrow-derived dendritic cells (BMDC) and splenic T lymphocytes by miR-574-5p or miR-574p-5p derivatives

About 1×10⁵ or 1×10⁶ mBMDCs from the wild-type mice or the B6. mTLR7^-/-mice were respectively seeded in 6-well plate and treated with 10 μg/ml of Dotap-PS-miR-574-5p or 100 ng/ml of LPS respectively. 24 h after the treatment, cells were harvested for flow cytometry analyses. Results showed that miR-574-5p significantly stimulated TNFαsecretion in CD11c⁺ BMDCs from the wild-type mice but in mTLR7 knockout mice the effects were greatly reduced (see FIG. 12a) .

About 1×10⁵ or 1×10⁶ splenic T lymphocyte from the wild-type mice or the B6. mTLR7^-/-mice were respectively seeded in 6-well plate and treated with 10 μg/ml of Dotap-PS-miR-574-5p or 10 μg/ml of Dotap-PS-miR-16 respectively. 24 h after the treatment, cells were harvested for flow cytometry analyses. Results showed thatmiR-574-5p significantly stimulated the activation (CD69) of splenic T lymphocyte from the wild-type mice but in mTLR7 knockout mice the effects were greatly reduced (see FIG. 12b) .

Example 12 Analyses of mRNA or protein expression in miR-574-5p-knocked-down HeLa cells by microarray hybridization, qPCR or Western blots

A volcano plot of mRNA expression in miR-574-5p knockdown HeLa cells was determined by NimbleGen 12x135K microarray hybridization (n ＝ 3) . Array hybridization and data processing were performed according to routine methods well known by those skilled in the art. The red point in the plot represents the differentially expressed genes with statistical significance. The vertical green lines in the plot divided genes that were up-regulated (151 genes) or down-regulated (661 genes) by at least 2 folds, respectively, whereas the horizontal green line represents a p-value of 0.05 (see FIG. 13a) . Protein expression of selected genes HLA-C, HLA-DRA, IRF8, and β-actin in miR-574-5p knockdown HeLa cells was determined by Western blots (see FIG. 13b) . mRNA expression of selected genes CCL2, CD74, HLA-DRA, HLA-C, IL6, IL8, IRF7, IRF8, NR4A1, OLR1, SFRS1, and TSC1 in miR-574-5p knockdown HeLa cells was determined by qPCR (n ＝ 3-5) (see FIG. 13c) . Enrichment of the 661 significantly down-regulated genes in miR-574-5p knockdown HeLa cells in 30 signaling or disease pathways (see FIG. 13d) . CCL2, (C-C Motif) ligand-2； CD74, cluster of differentiation-74； HLA-DRA, HLA class II histocompatibility antigen, DR alpha chain； HLA-C, major histocompatibility complex, Class I, C； IL8, interleukin-8； IRF8, interferon regulatory factor-8； NR4A1, nuclear receptor subfamily-4, group-A, member-1； OLR1, oxidized low-density lipoprotein receptor-1； SFRS1, serine/arginine-rich splicing factor-1； TSC1, tuberous sclerosis-1. NS, not significant； *, p < 0.05； **, p < 0.01； ***, p < 0.001, LV-miR-shRNA-ctrl versus LV-miR-574-5p-shRNA.

Example 13 Aberrant miR-574-5p signaling contributes significantly to cervical cancer development

Total RNAs from cultured cells or tissues were extracted using TRIzol according to the manufacturer’s protocols. Five microliters of total RNA was reverse transcribed using the ReverTra

Kit as instructed (TOYOBO, Shanghai, China) and miRNA-specific stem-loop primers listed in Table 4. qPCR was performed with total RNAs, using universal primer and miRNA-specific reverse LNA-primers as listed in Table 4, with U6 RNA served as an internal control.

MiR-574-5p expression in cervical cancer tissues and their adjacent normal tissues from 18 human patients was determined by qPCR. Data represented mean + SEM for three replicates (see FIG. 14a) . *, p < 0.05； **, p < 0.01； ***, p < 0.001, adjacent normal versus cervical tumor.

8-wk old male nude mice were subcutaneously inoculated with 2×10⁶ HeLa cells stably transduced with either LV-miR-shRNA-ctrl (left side) or LV-miR-574-5p-shRNA (right side) onto the dorsal flanks of animals. Athymic BALB/c nude mice were obtained from the SLAC Laboratory Animals Co Ltd, Shanghai, China. Tumors were dissected 4 wk after the inoculation. Results indicated that knocking-down of miR-574-5p greatly reduced tumor growth in the nude mice inoculated with HeLa cells (see FIG. 14b) .

Example 14 Measurements of miR-574-5p levels in human systemic lupus erythematosus (SLE) patients and female lupus-prone B6. Fas^lpr/^lpr mice

Serum total RNAs was extracted using a mirVana miRNA isolation kit (Cat#AM1556, Ambion, Austin, TX, USA) according to the manufacturer’s protocols whereas total RNAs from cultured cells or tissues were extracted using TRIzol according to the manufacturer’s protocols Five microliters of total RNA was reverse transcribed using the ReverTra

MiR-574-5p levels were measured by qPCR. FIG. 15a showed serum levels of miR-574-5p in normal healthy individuals and SLE patients as determined by qPCR. **, p < 0.01, normal healthy individuals versus SLE patients, n ＝ 11. FIG. 15b showed serum levels of miR-574-5p in the B6. WT or B6. Fas^lpr/lpr mice at ages of 90-d and 180-d as determined by qPCR. *, p < 0.05, ***, p < 0.001, B6. WT versus B6. Fas^lpr/lpr； n ＝ 3-6. FIG. 15c showed kidney levels of miR-574-5p in the B6. WT or B6. Fas^lpr/lpr mice at ages of 90-d and 180-d as determined by qPCR. *, p < 0.05, ***, p < 0.001, B6. WT versus B6. Fas^lpr/lpr； n ＝ 3-6. FIG. 15d showed miR-574-5p levels in the brain, heart, liver, lung, lymph node and spleen tissues of the B6. WT or B6. Fas^lpr/lprmice at the age of 90-d. NS, not significant； *, p < 0.05； B6. WT versus B6. Fas^lpr/lpr； n ＝ 6-7. FIG. 15e showed miR-574-5p levels in the brain, heart, liver, lung, lymph node and spleen tissues of the B6. WT or B6. Fas^lpr/lprmice at the age of 180-d. NS, not significant； *, p < 0.05, **, p < 0.01, B6. WT versus B6. Fas^lpr/lpr； n ＝ 6-7.

Together these data established that miR-574-5p is significantly up-regulated in the serum samples from human SLE patients and the serum and other tissues of female lupus-prone B6. Fas^lpr/^lpr mice.

Example 15 Knockdown of miR-574-5p significantly ameliorates SLE and lupus nephritis associated parameters in the B6. Fas^lpr/^lpr mice at the age of 20-wk

In vivo silencing of miR-574-5p was achieved by treatment with lentiviruses carrying shRNA against miR-574-5p, i.e. miR-574-5p inhibitor as described. FIG. 16a showed that lentivirus-mediated knockdown of miR-574-5p in the kidney and liver of B6. Fas^lpr/^lpr mice (n ＝ 6) . FIG. 16b showed inhibition of miR-574-5p significantly ameliorated lupus-associated splenomegaly (n ＝ 6) . FIG. 16c-h showed that silencing of miR-574-5p in the lupus-prone B6. Fas^lpr/lpr mice led to reduced serum anti-dsDNA autoantibody, blood urea nitrogen, proteinuria, serum TNFα, IL6 and IFNα (n ＝ 5-7) , although the differences for proteinuria and serum IFNα were not statistically significant. NS, not significant； **, p < 0.01； ***, p < 0.001, LV-miR-shRNA-ctrl versus LV-miR-574-5p-shRNA. Together these data established that knockdown of miR-574-5p significantly ameliorates SLE and lupus nephritis associated parameters in the B6. Fas^lpr/^lpr mice at the age of 20-wk.

Example 16 Histochemical and immunohistochemical staining analyses

Histochemical and immunohistochemical staining analyses of the renal and liver tissues in untreated B6. WT, LV-miR-shRNA-ctrl-treated B6. Fas^lpr/lpr and LV-miR-574-5p-shRNA treated B6. Fas^lpr/lprat the age of 20-wk were performed. Results were typical for at least three mice. FIG. 17a showed histochemical staining of the renal cortex by the PAS staining and immunohistochemistry staining of the renal cortex by anti-IgG antibody. FIG. 17b showed histochemical staining of the renal medulla by the PAS staining and immunohistochemistry staining of the renal medulla by anti-IgG antibody. FIG. 17c showed immunohistochemical staining of renal cortex by anti-CD68 antibody. FIG. 17d showed immunohistochemical staining of the liver tissues by anti-CD68 antibody.

Example 17 miR-574-5p knockdown altered distribution of splenic immune cells in the female B6. Fas^lpr/lpr mice

Splenic immune cells in B6. Fas^lpr/lpr at the age of 20-wk were treated with plasmids LV-miR-shRNA-ctrl or LV-miR-574-5p-shRNA respectively. Mouse spleen cell suspensions were prepared from the spleen tissues dissected from LV-miR-shRNA-ctrl-treated and LV-miR-574-5p-shRNA treated B6. Fas^lpr/lpr. After the elimination of red blood cells, spleen cells were stained with specific antibodies as indicated and used for flow cytometry analyses. T-helper cells, T_h (CD3⁺CD4⁺) ； cytotoxic T-cells, T_c (CD3⁺CD8⁺) ； natural killer cells, NK, (CD3^-NK1.1⁺) ； natural killer T-cells, NKT (CD3⁺NK1.1⁺) and regulatory T-cells, T_reg (CD4⁺CD25⁺) . NS, not significant； *, p < 0.05； LV-miR-shRNA-ctrl versus LV-miR-574-5p-shRNA treated； n ＝ 6 (see FIG. 18a) . Representative results for flow cytometry analyses of T_h and NK/NKT cells were shown in FIG. 18b. Representative results for flow cytometry analyses of T_c and T_reg cells were shown in FIG. 18c. Flow cytometry analyses showed altered distribution of splenic immune cells in the female B6. Fas^lpr/lpr mice being as a consequence of miR-574-5p knockdown.

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Claims

The use of miR-574-5p, miR-574-5p derivatives, and miR-574-5p inhibitors as immune modulators.
The use of claim 1, wherein the miR-574-5p and/or miR-574-5p derivatives are used as agonists for mammalian TLR7 and/or mammalian TLR8.
The use of claim 2, wherein the miR-574-5p and/or miR-574-5p derivatives are used as agonists for mTLR7 and/or hTLR8.
The use of any of claims 1 to 3, wherein the miR-574-5p derivatives are selected from the group consisting of PS-miR-574-5p, morpholino-miR-574-5p, 2’-O-methyl-miR-574-5p, 2’ -O-methoxyethyl-miR-574-5p, 2’ -fluoro-miR-574-5p, and combinations thereof.
The use of claim 1, wherein the miR-574-5p inhibitors are used as antagonists for mammalian TLR7 and/or mammalian TLR8.
The use of claim 1 or 5, wherein the miR-574-5p inhibitors are used as antagonists for mTLR7 and/or hTLR8.
The use of claim 1, 5 or 6, wherein the miR-574-5p inhibitors are selected from the group consisting of shRNA against miR-574-5p, single-stranded RNA complementary to miR-574-5p, double-stranded siRNA targeting miR-574-5p, single-stranded DNA complementary to miR-574-5p； the derivatives of the single-stranded RNA complementary to miR-574-5p, double-stranded siRNA targeting miR-574-5p, and single-stranded DNA complementary to miR-574-5p in the forms of phosphorothiolate modification, morpholino modification, 2’ -O-methyl-modification, 2’ -O-methoxyethyl-modification, 2’-fluoro-modification, LNA-modification； or combinations thereof.
A composition for inducing mammalian TLR7 and/or mammalian TLR8 mediated immune responses in cells or a subject, containing miR-574-5p, miR-574-5p derivatives or combinations thereof, and/or pharmaceutically acceptable excipients.
The composition of claim 8, wherein the miR-574-5p derivatives are selected from the group consisting of PS-miR-574-5p, morpholino-miR-574-5p, 2’ -O-methyl-miR-574-5p, 2’-O-methoxyethyl-miR-574-5p, 2’ -fluoro-miR-574-5p, and combinations thereof.
The composition of claim 8, wherein the mammalian TLR7 is mTLR7, and the mammalian TLR8 is hTLR8.
A method for inducing mammalian TLR7 and/or mammalian TLR8 mediated immune responses in a subject, comprising administering effective amount of miR-574-5p, miR-574-5p derivatives, or combinations thereof to a subject in need thereof.
The method of claim 11, wherein the miR-574-5p derivatives are selected from the group consisting of PS-miR-574-5p, morpholino-miR-574-5p, 2’ -O-methyl-miR-574-5p, 2’-O-methoxyethyl-miR-574-5p, 2’ -fluoro-miR-574-5p, and combinations thereof.
The method of claim 11, wherein the mammalian TLR7 is mTLR7, and the mammalian TLR8 is hTLR8.
A method for inducing mammalian TLR7 and/or mammalian TLR8 mediated immune responses in a subject, comprising administering the composition of claim 5 or 6 in an effective amount to a subject in need thereof.
The method of claim 14, wherein the mammalian TLR7 is mTLR7, and the mammalian TLR8 is hTLR8.
A composition for inhibiting mammalian TLR7 and/or mammalian TLR8 mediated immune responses, containing the miR-574-5p inhibitors and/or pharmaceutically acceptable excipients.
The composition of claim 16, wherein the miR-574-5p inhibitors are selected from the group consisting of shRNA against miR-574-5p, single-stranded RNA complementary to miR-574-5p, double-stranded siRNA targeting miR-574-5p, single-stranded DNA complementary to miR-574-5p； the derivatives of the single-stranded RNA complementary to miR-574-5p, double-stranded siRNA targeting miR-574-5p, and single-stranded DNA complementary to miR-574-5p in the forms of phosphorothiolate modification, morpholino modification, 2’ -O-methyl-modification, 2’ -O-methoxyethyl-modification, 2’-fluoro-modification, LNA-modification； or combinations thereof.
The composition of claim 16, wherein the mammalian TLR7 is mTLR7, and the mammalian TLR8 is hTLR8.
A method for inhibiting mammalian TLR7 and/or mammalian TLR8 mediated immune responses in a subject, comprising administering effective amount of miR-574-5p inhibitors to a subject in need thereof.
The method of claim 19, wherein the miR-574-5p inhibitors are selected from the group consisting of shRNA against miR-574-5p, single-stranded RNA complementary to miR-574-5p, double-stranded siRNA targeting miR-574-5p, single-stranded DNA complementary to miR-574-5p； the derivatives of the single-stranded RNA complementary to miR-574-5p, double-stranded siRNA targeting miR-574-5p, and single-stranded DNA complementary to miR-574-5p in the forms of phosphorothiolate modification, morpholino modification, 2’ -O-methyl-modification, 2’ -O-methoxyethyl-modification, 2’-fluoro-modification, LNA-modification； or combinations thereof.
The method of claim 19, wherein the mammalian TLR7 is mTLR7, and the mammalian TLR8 is hTLR8.
A method for inhibiting mammalian TLR7 and/or mammalian TLR8 mediated immune responses in a subject, comprising administering composition of claim 16 or 17 in an effective amount to a subject in need thereof.
The method of claim 22, wherein the mammalian TLR7 is mTLR7, and the mammalian TLR8 is hTLR8.
Use of miR-574-5p, miR-574-5p derivatives, or combinations thereof as adjuvant.
Use of claim 24, wherein the adjuvant is administered with a vaccine, an antibacterial agent, or an antigen.
The use of claim 24, wherein the miR-574-5p derivatives are selected from the group consisting of PS-miR-574-5p, morpholino-miR-574-5p, 2’ -O-methyl-miR-574-5p, 2’-O-methoxyethyl-miR-574-5p, 2’ -fluoro-miR-574-5p, and combinations thereof.
A composition containing miR-574-5p, miR-574-5p derivatives, or combinations thereof in an effective amount, and a vaccine, an antibacterial agent, or an antigen, as well as pharmaceutically acceptable excipients.
The composition of claim 27, wherein the miR-574-5p derivatives are selected from the group consisting of PS-miR-574-5p, morpholino-miR-574-5p, 2’ -O-methyl-miR-574-5p, 2’-O-methoxyethyl-miR-574-5p, 2’ -fluoro-miR-574-5p, and combinations thereof.
A method for treating a subject having diseases or disorders related to mammalian TLR7 and/or mammalian TLR8 signaling, comprising administering to the patient having such disorders or diseases effective amount of miR-574-5p, miR-574-5p derivatives, or miR-574-5p inhibitors, wherein the diseases or disorders related to mammalian TLR7 and/or mammalian TLR8 signaling are selected from the group consisting of cancer, autoimmune disorders, airway inflammation, inflammatory disorders, infectious diseases, skin disorders, allergy, asthma or diseases caused by pathogens.
The method of claim 29, wherein the miR-574-5p derivatives are selected from the group consisting of PS-miR-574-5p, morpholino-miR-574-5p, 2’ -O-methyl-miR-574-5p, 2’-O-methoxyethyl-miR-574-5p, 2’ -fluoro-miR-574-5p, and combinations thereof； and wherein the miR-574-5p inhibitors are selected from the group consisting of shRNA against miR-574-5p, single-stranded RNA complementary to miR-574-5p, double-stranded siRNA targeting miR-574-5p, single-stranded DNA complementary to miR-574-5p； the derivatives of the single-stranded RNA complementary to miR-574-5p, double-stranded siRNA targeting miR-574-5p, and single-stranded DNA complementary to miR-574-5p in the forms of phosphorothiolate modification, morpholino modification, 2’-O-methyl-modification, 2’ -O-methoxyethyl-modification, 2’-fluoro-modification, LNA-modification； or combinations thereof.
The method of claim 29, wherein the mammalian TLR7 is mTLR7, and the mammalian TLR8 is hTLR8.
A method for preventing diseases or disorders related to mammalian TLR7 and/or mammalian TLR8 signaling from a subject, comprising administering to the patient having such a disorder or disease miR-574-5p, miR-574-5p derivatives, or miR-574-5p inhibitors in a therapeutically effective amount, wherein the diseases or disorders related to mammalian TLR7 and/or mammalian TLR8 signaling are selected from the group consisting of cancer, autoimmune disorders, airway inflammation, inflammatory disorders, infectious diseases, skin disorders, allergy, asthma or diseases caused by pathogens.
The method of claim 32, wherein the miR-574-5p derivatives are selected from the group consisting of PS-miR-574-5p, morpholino-miR-574-5p, 2’ -O-methyl-miR-574-5p, 2’-O-methoxyethyl-miR-574-5p, 2’ -fluoro-miR-574-5p, and combinations thereof； and wherein the miR-574-5p inhibitors are selected from the group consisting of shRNA against miR-574-5p, single-stranded RNA complementary to miR-574-5p, double-stranded siRNA targeting miR-574-5p, single-stranded DNA complementary to miR-574-5p； the derivatives of the single-stranded RNA complementary to miR-574-5p, double-stranded siRNA targeting miR-574-5p, and single-stranded DNA complementary to miR-574-5p in the forms of phosphorothiolate modification, morpholino modification, 2’-O-methyl-modification, 2’ -O-methoxyethyl-modification, 2’-fluoro-modification, LNA-modification； or combinations thereof.
The method of claim 32, wherein the mammalian TLR7 is mTLR7, and the mammalian TLR8 is hTLR8.
The method of claim 29 or 32, the diseases or disorders related to mammalian TLR7 and/or mammalian TLR8 signaling are selected from the group consisting of autoimmune diseases and inflammatory disorders such as SLE, MS, and asthma； viral infection such as H5N1, VSV, and SARS； bacterial infection such as sepsis； graft versus host diseases； cancer such as lung cancer, pancreatic cancer, CRC, prostate cancer, and HNC； and cardiovascular and pulmonary arterial hypertension.
The method of claim 35, wherein the mammalian TLR7 is mTLR7, and the mammalian TLR8 is hTLR8.
A method of diagnosing risks related to immune-related conditions in a subject, comprising: (i) identifying the relative miR-574-5p expression compared to control, and (ii) diagnosing increased risk of immune related conditions in the subject if the subject has increased miR-574-5p expression compared to control, or (iii) diagnosing no increased risk of immune related conditions in the subject if the subject does not have increased miR-574-5p expression compared to control.
The use of miR-574-5p, miR-574-5p derivatives, or miR-574-5p inhibitors in the preparation of drugs for treating diseases or disorders related to mammalian TLR7 and/or mammalian TLR8 signaling.
The use of claim 38, wherein the miR-574-5p derivatives are selected from the group consisting of PS-miR-574-5p, morpholino-miR-574-5p, 2’ -O-methyl-miR-574-5p, 2’-O-methoxyethyl-miR-574-5p, 2’ -fluoro-miR-574-5p, and combinations thereof； and wherein the miR-574-5p inhibitors are selected from the group consisting of shRNA against miR-574-5p, single-stranded RNA complementary to miR-574-5p, double-stranded siRNA targeting miR-574-5p, single-stranded DNA complementary to miR-574-5p； the derivatives of the single-stranded RNA complementary to miR-574-5p, double-stranded siRNA targeting miR-574-5p, and single-stranded DNA complementary to miR-574-5p in the forms of phosphorothiolate modification, morpholino modification, 2’-O-methyl-modification, 2’ -O-methoxyethyl-modification, 2’-fluoro-modification, LNA-modification； or combinations thereof.
The use of claim 38, wherein the mammalian TLR7 is mTLR7, and the mammalian TLR8 is hTLR8.