CN110585432B - Application of fibroblast growth factor receptor 1in preparation of medicine for preventing and treating enterovirus 71infection - Google Patents
Application of fibroblast growth factor receptor 1in preparation of medicine for preventing and treating enterovirus 71infection Download PDFInfo
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Abstract
The invention relates to the technical field of biomedicine, in particular to a new target for resisting enterovirus 71infection and application thereof. The invention takes human colon cancer cells (Caco-2) as target cells, and adopts RNA interference technology to down-regulate the expression of target cell membrane receptors, so as to search host factors capable of effectively inhibiting EV71 from infecting the human colon cancer cells, thereby achieving the purpose of blocking EV71 from the source (intestinal tract). Experiments prove that fibroblast growth factor receptor 1(FGFR1) plays an important role in EV71 infected Caco-2 cells, expression of FGFR 1is down-regulated, and infection of EV71 can be obviously inhibited. The invention provides application of FGFR 1in preparation of a medicament for preventing or treating enterovirus 71infection, and provides a new target and a treatment scheme for clinically preventing and treating diseases such as hand-foot-and-mouth disease, meningitis, brainstem encephalitis or poliomyelitis caused by EV71 infection.
Description
Technical Field
The invention relates to the technical field of biomedicine, in particular to a new target for resisting enterovirus 71infection and application thereof.
Background
Enterovirus 71 (Enterovirus 71, EV71) belongs to human enterovirus A of Enterovirus of picornaviridae, and is one of the main pathogens causing Hand-foot-and-mouth disease (HFMD). Currently, hand-foot-and-mouth disease is fulminant and prevalent in many areas of the world, especially in the asia-pacific region. The main disease population of the hand-foot-and-mouth disease is infants under 5 years old, and the clinical manifestations are fever, herpes and other symptoms of hands, feet, buttocks, oral mucosa and other parts; a small number of children may develop into critically ill patients with Central Nervous System (CNS) lesions, including aseptic meningitis, brainstem encephalitis, encephalomyelitis, and neurogenic pulmonary edema, that present a serious threat to the life and health of the infant (Solomon T, et al. virology, epidemiology, pathobiology, and control of infectious 71.Lancet infection dis.2010,10(11): 778-90). Hand-foot-and-mouth disease, especially severe cases, is mostly caused by enterovirus 71 (EV71) infection. However, at present, no specific and efficient antiviral drug exists for treating hand-foot-and-mouth disease caused by EV71, symptomatic treatment is mainly adopted clinically, and no effective vaccine appears in the aspect of prevention.
The transmission of EV71 is most widespread in the faecal-oral route, where the intestinal tract is the first barrier of the human body against pathogens. Animal experimental studies have demonstrated that EV71 is pathogen positive initially in small intestine tissue after oral inoculation of mice [ Chen YC, et al. A Murine oral enterovirus 71infection model with central nervous system in vivo. journal of General Virology,2004, 85(1):69-77 ]. Thus, EV71 first replicates rapidly in large numbers in small intestinal cells by infecting the intestinal system, and then enters the blood causing viremia and causing infection of other organs. However, the mechanism of how the virus invades the intestinal cells after EV71 reaches the human intestinal system is not clear. The small intestine is used as an important organ of a human digestive system and is mainly responsible for digestion and absorption of nutrients, and the outermost layer of the cavity surface of the small intestine is small intestine mucosal epithelial cells which play an important role in resisting pathogens. As a human colon cancer cell line, Caco-2 is similar to human small intestine mucosal epithelial cells in morphological and physiological functions, and the cultured mature Caco-2 is a compact monolayer cell with the same polarity and close connection and microvilli structure as normal small intestine mucosal epithelial cells; and as tumor cells, are easier to culture than small intestine mucosal epithelial cells. Therefore, the mechanism of detecting EV71 infection Caco-2 and finding a new antiviral target by the mechanism are important for preventing EV71 infection. However, the specific molecular mechanism of EV71 infection with Caco-2 is still unclear.
When a virus infects a host, it must utilize various molecules and related signaling pathways of the host cell to complete its life cycle. Among other things, cytokine receptors have been found to play an important role in the infection of host cells by viruses and in the process of replication within host cells. For example, the C-C chemokine receptor type 5 (CCR5) has been the focus of intensive research since its discovery in 1996 as a role of an HIV entry co-receptor, driving the development of small molecule drugs directed to CCR 5. In 2007, maraviroc was the first clinically approved chemokine receptor inhibitor for the treatment of HIV [ Brelot A, et al. CCR5Revisited: How Mechanisms of HIV Entry Govern AIDS Patholonesis. J Mol biol.2018,430(17):2557-2589 ]. In recent years, platelet-derived growth factor receptor alpha (PDGFR-alpha) has also been found as a receptor for adeno-associated virus type 5 and human cytomegalovirus to mediate viral invasion [ Di plasmid G, et al.identification of PDGFR as a receptor for AAV-5 transmission. Nat. med.2003,9(10): 1306-12; wu Y, et al, human cytomegavirus glycoprotein complex gH/gL/gO uses PDGFR-alpha as a key for entry, PLoS Patholog.2017, 13(4) e 1006281. It has also been shown that the Hepatitis B virus X protein promotes the proliferation and migration of Hepatitis B virus-associated hepatoma cells by up-regulating the expression of the interleukin-7receptor (IL7R) [ Kong F, et al, Hepatitis B virus X protein promoters internemeukin-7 receptor expression via NF-. kappa.B and Notch1pathway to failure promotion and migration of Hepatitis B virus-related hepatoma cells J Exp Clin Cancer Res.2016, (35) (172) ]. The up-regulation of IL7R has also been reported to promote the early stages of HIV infection [ Zhang M, et al. HIV regulation of the IL-7R: a viral mechanism for enhancing HIV-1replication in human mammophages in vitro. J. Leukoc biol.2006,79(6):1328-38 ]. In addition, it has been found that the knock-down of the interleukin 10 receptor (IL10R) can significantly inhibit the replication of porcine reproductive and respiratory syndrome virus [ Chen Y, et al, knock-down expression of IL-10R α gene inhibitors PRRSV replication and elevoses immunity in PBMCs of Tibeta pig in vitro, vet Res Commun.2018, 42(1):11-18 ]. Therefore, cytokine receptors have become important targets for antiviral drug screening.
The interleukin 4receptor (IL4R) is a type I transmembrane protein. It is widely expressed in various tissues and organs throughout the body, and is highly enriched in immune organs such as appendix, spleen, lymph node, bone marrow and tonsil, and tissues and organs such as lung and colon [ https:// www.proteinatlas.org/ENGG 00000077238-IL4R/tissue ]. IL4R can exert various immunoregulatory effects after binding with ligands such as interleukin 4 and interleukin 3, such as regulating IgE antibody production in B cells, promoting differentiation of Th2 cells, and inducing macrophage replacement activation. With the progress of the study, it was found that neonatal mouse lung lesions caused by Respiratory Syncytial Virus (RSV) and Rhinovirus (RV) -induced airway mucus metaplasia can be significantly inhibited by inhibiting IL4R expression or knocking out IL4R [ Ripple MJ, et al.immunization with IL4R alpha antisense oligonucleotide precursors respiratory disease. j immune. 2010, 185(8): 4804-11; schneider D, et al, New, fungal infection indexes, and airborne hyperresponsiveness. J Immunol.2012,188(6):2894-904 ]. It has also been shown that the HIV type 1tat gene can maintain its own replication by promoting transcriptional upregulation of IL4R [ Husain SR, et al. transcriptional up-regulation of interleukin 4receptors by human immunodeficiency virus type 1tat gene. AIDS Res Hum retroviruses.1996, 12(14):1349-59 ]. These results all indicate that IL4R plays an important role in the pathogenic process of various viruses.
Fibroblast growth factor receptor 1(FGFR1), also known as basic Fibroblast growth factor receptor 1, fms-related tyrosine kinase-2 or CD 331, belongs to a Fibroblast growth factor receptor family member (including FGFR2, FGFR3, FGFR4 and FGFRL 1in addition to FGFR 1). FGFR 1is a cell surface membrane receptor with tyrosine kinase activity, the ligand of which is a specific member of the fibroblast growth factor family. FGFR1 participate in a variety of biological processes by activating signaling pathways to elicit a cellular response when bound to a suitable ligand, such as FGF 1. Research finds that FGFR1 plays an important role in infection and pathogenesis of various viruses. For example, FGFR1 can act as a co-receptor for adeno-associated virus 2(AAV2) and adeno-associated virus 3(AAV3), mediating infection of host cells by AAV2 [ Qing K, et al human fibroblast growth promoter 1is a co-receptor for infection by vector-assisted virus 2.Nat med.1999,5(1): 71-7; black burn SD, et al, Attachment of amplified virus type 3H to fiber growth factor receiver 1.Arch Virus.2006, 151(3):617-23 ]. Epstein-Barr Virus (EBV) encoded latent membrane protein 1(LMP1) promotes aerobic glycolysis and transformation of human nasopharyngeal epithelial cells by increasing FGFR1 expression and mediating constitutive activation of the FGFR1 signaling pathway, thereby participating in the pathogenesis of non-keratinized nasopharyngeal carcinoma [ Lo AK, et al.activation of the FGFR1signalling pathway by the Epstein-Barr virus-encoded LMP1 proteins involved in physiological glycosylation and transformation of human nasopharyngeal carcinoma J Pathol.2015, 237(2):238-48 ]. However, it has been shown that FGFR1 can be used as a mechanism of host cells for resisting replication of Influenza A Virus, siRNA interfering FGFR1 can promote replication of Influenza A/PR 8 and H5N1 Virus in A549 cells, and lentivirus-mediated exogenous FGFR1 expression can significantly inhibit replication of Influenza A Virus [ Liu X, et al. A Functional Role of Fibroplast Growth Factor Receptor 1(FGFR1) in the above expression of Influenza A Virus replication.
However, at present, no report about the effects of IL4R and FGFR1 molecules in enterovirus, particularly EV71 infected host cells exists, and intensive research on the two molecules can not only improve the understanding of EV71 infection and pathogenesis, but also provide a new idea and target point for preventing and treating EV71 infection.
Disclosure of Invention
The invention aims to provide a new target for resisting enterovirus 71infection, namely Fibroblast growth factor receptor 1(FGFR 1).
Another object of the present invention is to provide a novel use of the fibroblast growth factor receptor 1(FGFR1) molecule, in particular, in the treatment of enterovirus type 71 infection.
The third purpose of the invention is to provide siRNA interfering the molecular expression of fibroblast growth factor receptor 1(FGFR1) and application thereof.
In order to achieve the purpose, the main technical scheme of the invention is as follows:
according to the invention, a human colon cancer cell (Caco-2) is taken as a target cell, and the expression of a target cell cytokine receptor is regulated down by adopting an RNA interference technology, so that a host factor capable of effectively inhibiting EV71 from infecting the human colon cancer cell (Caco-2) is searched, and the purpose of blocking EV71 infection from a source (intestinal tract) is achieved. The invention selects a group of host cell cytokine receptors for screening, and the molecules mainly distributed on cell membranes play an important role in signal transduction of host cells and are molecules which are easy to be hijacked and utilized by viruses in the virus infection process. These molecules include: bone morphogenetic protein receptor type 2 (BMPR2), C-C chemokine receptor type 5 (CCR5), C-C chemokine receptor type 7 (CCR7), fibroblast growth factor receptor 1(FGFR1), interleukin 17 receptor B (IL17RB), interleukin 1 receptor 1(IL1R1), interleukin 4receptor (IL4R), platelet-derived growth factor receptor a (pdgfra), Toll-like receptor 2(TLR2), and tumor necrosis factor receptor superfamily member 11B (TNFRSF 11B). The effect on EV71 infection was observed by retrieving the complete sequence and mRNA sequence from NCBI GeneBank, performing biological analysis on these genes using existing network resources and common software, selecting the coding region as the target sequence for siRNA design, then designing siRNA, and down-regulating these molecules.
Experiments prove that fibroblast growth factor receptor 1(FGFR1) plays an important role in EV71 infected Caco-2 cells, expression of FGFR 1is down-regulated, and infection of EV71 can be obviously inhibited.
Based on this, in the first aspect of the invention, a new target against enterovirus type 71infection, namely fibroblast growth factor receptor 1(FGFR1), is provided.
In a second aspect of the invention, the invention provides an application of fibroblast growth factor receptor 1(FGFR1) in preparing a medicament for preventing or treating enterovirus 71 infection.
Furthermore, the application refers to the fibroblast growth factor receptor 1(FGFR1) serving as an intervention target for preventing or treating enterovirus 71 infection.
Further, the diseases caused by the enterovirus 71infection include, but are not limited to, hand-foot-and-mouth disease, meningitis, brainstem encephalitis, poliomyelitis and the like. That is, the present invention also provides an application of fibroblast growth factor receptor 1(FGFR1) in the preparation of a medicament for preventing or treating diseases including, but not limited to, hand-foot-mouth disease, meningitis, brainstem encephalitis, polio, and the like, caused by enterovirus type 71 infection.
Furthermore, the medicine is used for inhibiting enterovirus 71 type infection by inhibiting or down-regulating the expression level of fibroblast growth factor receptor 1(FGFR 1).
In a third aspect of the invention, the application of an agent for inhibiting or down-regulating the expression level of fibroblast growth factor receptor 1(FGFR1) in preparing a medicament for preventing or treating enterovirus 71infection is provided.
Further, the reagent for inhibiting or down-regulating the expression level of FGFR1 refers to siRNA, shRNA, miRNA, or antisense nucleotide that specifically interferes with FGFR1 gene expression, processing, or a recombinant vector (such as a plasmid) containing siRNA, shRNA, miRNA, or antisense nucleotide.
In one embodiment of the invention, the agent for inhibiting or down-regulating the expression level of FGFR 1is interfering RNA (sirna) of fibroblast growth factor receptor 1, wherein the sequence of the interfering RNA is selected from any one of the following:
GUAGCAACGUGGAGUUCAU(SEQ ID NO:10)
GCAAGAUUGGCCCAGACAA(SEQ ID NO:11)
GGUCGGUCAUCGUCUACAA(SEQ ID NO:12)
wherein, the siRNA shown in SEQ ID NO. 10 has the best effect of down-regulating the expression level of FGFR1, and the infection of EV71 to Caco-2 cells is most obviously reduced.
In a fourth aspect, the present invention provides a medicament for preventing or treating enterovirus 71infection, the medicament comprising an agent that inhibits or down-regulates the expression level of FGFR 1.
The invention has the advantages that:
the invention screens out a novel host cell molecule FGFR1 capable of inhibiting EV71 from infecting Caco-2 cells. After the FGFR1 gene is down-regulated, the normal physiological function of cells is not influenced, but the infection of the EV71 to Caco-2 cells is obviously inhibited. Therefore, the invention provides a new target point and a treatment scheme for clinically preventing and treating the hand-foot-and-mouth disease, the nervous system and the cardio-pulmonary system diseases caused by the EV71 infection.
Drawings
FIG. 1is a graph showing the relative expression level and cytotoxicity of a target gene after transfection of effective siRNA, in which the major axis (left y axis) represents the relative expression level (expressed as RQ value) of the target gene detected by real-time fluorescent quantitative PCR (RT-PCR) method, and the minor axis (right y axis) represents the toxicity of each siRNA transfected into cells detected by CCK-8 kit (expressed as the cell survival rate from the normalization to the idling control group);
CTRL: caco-2 cell group without any siRNA transfection (free-run control group);
siRNA: caco-2 cell group transfected with siRNA against each target gene (experimental group);
*：p<0.05。
FIG. 2 is a graph of immunofluorescence to detect the effect of each transport-associated membrane protein on EV71 infection after being down-regulated, wherein A is a fluorescence observation graph of virus infectivity after each protein is down-regulated, and B is a statistical graph of virus infection rate relative to an empty transfer control group after each molecule is down-regulated;
CTRL: caco-2 cell group without any siRNA transfection (free-run control group);
NT: a Caco-2 cell group (negative control group) transfected with non-targeting siRNA;
siRNA: caco-2 cell group (Experimental group) transfected with siRNA against each target Gene
Horizontal dotted line: reference line for 50% virus infection rate relative to the idling control group.
FIG. 3 shows that after transfection of siRNA targeting FGFR1(A) and IL4R (B) with different sequences, real-time fluorescence quantitative PCR (RT-PCR) method is used to detect mRNA level of target gene (expressed by RQ value of relative expression of FGFR1 and IL4R gene);
CTRL: caco-2 cell group without any siRNA transfection (free-run control group);
NT: a Caco-2 cell group (negative control group) transfected with non-targeting siRNA;
FGFR 1-10: a Caco-2 cell group transfected with siRNA (SEQ ID NO:10) against FGFR1 gene;
FGFR 1-11: a Caco-2 cell group transfected with siRNA (SEQ ID NO:11) against FGFR1 gene;
FGFR 1-12: a Caco-2 cell group transfected with siRNA (SEQ ID NO:12) against FGFR1 gene;
IL 4R-19: a Caco-2 cell group transfected with siRNA (SEQ ID NO:19) against IL4R gene;
IL 4R-20: a Caco-2 cell group transfected with siRNA (SEQ ID NO:20) against the IL4R gene;
IL 4R-21: a Caco-2 cell group transfected with siRNA (SEQ ID NO:21) against IL4R gene;
*：p<0.05。
FIG. 4 shows the effect of down-regulation of FGFR1 and IL4R on EV71 infection, wherein A and B are viral RNA loads (expressed as the relative expression RQ value of EV71 RNA) detected by RT-PCR method, and C and D are viral protein expression detected by Western Blot method;
CTRL: caco-2 cell group without any siRNA transfection (empty cell group);
NT: a Caco-2 cell group (negative control group) transfected with non-targeting siRNA;
FGFR 1-10: a Caco-2 cell group transfected with siRNA (SEQ ID NO:10) against FGFR1 gene;
FGFR 1-11: a Caco-2 cell group transfected with siRNA (SEQ ID NO:11) against FGFR1 gene;
FGFR 1-12: a Caco-2 cell group transfected with siRNA (SEQ ID NO:12) against FGFR1 gene;
IL 4R-19: a Caco-2 cell group transfected with siRNA (SEQ ID NO:19) against IL4R gene;
IL 4R-20: a Caco-2 cell group transfected with siRNA (SEQ ID NO:20) against the IL4R gene;
IL 4R-21: a Caco-2 cell group transfected with siRNA (SEQ ID NO:21) against IL4R gene;
GAPDH: an endogenous reference protein glyceraldehyde-3-phosphate dehydrogenase (36 kDa);
*：p<0.05。
Detailed Description
The following examples are provided to illustrate specific embodiments of the present invention.
The reagents and starting materials used in the present invention are commercially available or can be prepared according to literature procedures. Experimental procedures without specific conditions noted in the following examples, generally following conventional conditions such as Sambrook et al molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), either according to conventional conditions or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are by weight.
Example 1:
1, designing and synthesizing specific siRNA sequence of each transport-associated membrane protein.
1.1 aiming at each target gene, NCBI GeneBank is searched to obtain the complete sequence and mRNA sequence, the AXIIR is biologically analyzed by utilizing the existing network resources and common software, and the coding region is selected as the target sequence for siRNA design. Referring to the siRNA design principle, comparing the blast function of a GeneBank database with a human genome sequence to ensure no homology; potential siRNA of pairing 8 continuous basic groups at the 5' end of the aitisense chain with other genes is excluded; any potential siRNA with a stretch of 14 consecutive bases pairing with other genes is excluded. And the design software is used for pre-evaluation and determination, 3 optimal dynamic parameter targets are selected to enter the subsequent experimental process, and each gene synthesizes 3 interference sequences in total, which is shown in table 1.
1.2 Synthesis and purification of Single-stranded siRNA was accomplished by Invitrogen.
TABLE 1 design of siRNA targets
2siRNA sequence screening and interference effect identification
2.1RNA transfection
The transfection procedure was performed according to the instruction of DharmaFECT-1 (available from Horizon, cat. No. T-2001-02)
1) Caco-2 cells (purchased from ATCC under the designation HTB-37) at 3X 104The cells were seeded in 24-well cell culture plates, cultured at 37 ℃ and transfected with siRNA at a cell density of about 70%.
2) Adding 4. mu.L DharmaFECT-1 into 46. mu.L opti-MEM, mixing, and incubating at room temperature for 5 min; another 5. mu.L of siRNA at a concentration of 5. mu.M was mixed with 46. mu.L of opti-MEM. After incubation, diluted DharmaFECT-1 transfection reagent was added to the diluted siRNA and gently pipetted and mixed. After incubation at room temperature for 15min, the cells were added to Caco-2 cells and supplemented with 400. mu.L of opti-MEM to give a final RNA concentration of 50 nM.
3) DMEM medium (purchased from Thermo Fisher scientific, Inc., Cat. 12430062) containing 10% fetal bovine serum (purchased from Thermo Fisher scientific, Inc., Cat. 10437028) was replaced at 6-8 hours.
2.2 real-time fluorescent quantitative PCR (RT-PCR) detection of mRNA levels of respective target proteins
1) Extracting total RNA of each group of cells, and the specific steps are as follows:
after 48 hours of transfection, the culture supernatant was removed, and 1ml of RNAasso Plus (purchased from TAKARA, cat # 9109) was added to the cells, and the cells were lysed on ice with thorough mixing for 5-10 minutes. The mixture was transferred to an EP tube, 1/5 volumes of chloroform were added, and shaking vigorously for 15 seconds. Centrifuge at 12,000 rpm for 15 minutes at 4 ℃. The upper aqueous phase was transferred to a new EP tube, an equal volume of isopropanol was added, mixed well and allowed to stand at room temperature for 10 minutes. Centrifuge at 12,000 rpm for 10 minutes at 4 ℃. The supernatant was discarded and 1ml of ice-cold 75% ethanol was added. Centrifuge at 12,000 rpm for 10 minutes at 4 ℃. The supernatant was discarded sufficiently, the RNA precipitate was air-dried at room temperature, and RNase-Free dH (RNase Free dH) was added2O) dissolving the precipitate to obtain total RNA.
2) Total cDNA was prepared using a reverse transcription kit (purchased from TAKARA, Inc., cat # RR036A) by the following specific steps:
the following reaction system was added to the PCR tube,
5×PrimeScript RT Master Mix 2μL
Total RNA 500ng
Rnase Free dH2O up to 10μL
the mixture was gently mixed and mixed, and the mixture was reacted at 37 ℃ for 15 minutes (reverse transcription reaction) and then inactivated at 85 ℃ for 5 seconds (reverse transcriptase inactivation reaction).
3) RT-PCR detection of target gene expression level
The reaction was carried out using TB Green Premix Ex Taq kit (purchased from TAKARA, Inc., cat # RR420A) in the following reaction system,
two-step amplification was performed using an Applied Biosystems 7300plus instrument:
4) and calculating the relative expression quantity of each target gene by adopting a delta t method.
RQ＝2-ΔΔt＝2- [ (Ct treatment sample target Gene-Ct control sample target Gene) - (Ct treatment sample internal reference Gene-Ct control sample internal reference Gene)]
Interference efficiency is 1-RQ
3 cytotoxicity assay
The CCK-8 method is adopted to detect the influence of the transfected siRNA on cell proliferation, and the specific steps are as follows:
cells in the logarithmic growth phase were collected and seeded at a density of 3000 per well in 96-well plates. Cells were cultured to about 70% confluence, each siRNA was transfected, cultured for 48 hours, the original medium was discarded, 100. mu.L of a fresh medium containing 10. mu.L of CCK-8 reagent (purchased from Nippon college chemical research institute, cat # CK04) was added to each well, and absorbance at a wavelength of 450nm was measured in each well using a multifunctional microplate reader after 3 to 4 hours of culture. The experiments were independently repeated 3 times, mean values were calculated, and results were normalized to the idle control group.
4EV71 virus infection Caco-2 cell
4.1 experiment of EV71 Virus infection with Caco-2 cells
The EV71 virus infection experiment is carried out 48 hours after Caco-2 cells are transfected with siRNA. The culture supernatant was aspirated, rinsed 2 times with pre-warmed PBS, EV71 was inoculated with a virus amount of MOI 0.1, incubated at 37 ℃ for 2 hours, the virus solution was discarded, rinsed 3 times with pre-warmed PBS, and the culture was continued with addition of fresh medium.
4.2 immunofluorescence staining detection of EV71 antigen expression
The Caco-2 cells are continuously cultured for 48h after being infected with the virus, and the expression of the virus antigen is detected by adopting an immunofluorescence method, and the method comprises the following specific steps:
1) cell fixation and membrane penetration: the culture medium in the 96-well plate was removed, PBS was added to wash the cells for 2 times, 100. mu.l of pre-cooled methanol (both fixation and membrane permeation) was added to each well, the membrane permeation was fixed at-20 ℃ for 20min, and the cells were washed with pre-cooled PBS for 3 times.
2) And (3) sealing: mu.l of 3% BSA was added to each well and incubated for 1h at room temperature.
3) Primary antibody incubation: 100. mu.l of EV 71-specific murine mAb 10F0(1:1000 dilution) was added to each well, incubated overnight at 4 ℃ in a shaker, and washed 3 times with pre-cooled PBS.
4) And (3) secondary antibody incubation: 100. mu.l of AF 488 fluorescence-labeled anti-mouse IgG (diluted 1: 1000) was added to each well, incubated at room temperature for 1h in the dark, and washed 3 times with pre-cooled PBS in the dark.
5) Marking cell nucleus: cell nucleus fluorescent dye DAPI (1:10000, PBS dilution) was added to each well, incubated at room temperature in the dark for 15min, and washed 3 times with pre-cooled PBS in the dark.
6) The green AF 488-positive cell rate was detected and calculated under a fluorescence microscope.
4.3 Western blot.
(1) And respectively extracting the total protein of each group of Caco-2 cells by using protein lysate.
(2) After protein quantification, 20ug of protein was added to 10% SDA-PAGE gels, and the corresponding bands were removed and transferred to PVDF membrane using an electrotransfer.
(3) Non-specific sites of the protein were blocked with 5% skim milk, incubated with diluted EV 71-specific murine mAb 10F0(1:1000 dilution) overnight at 4 ℃ and rinsed three times with TBST buffer.
(4) The cells were then incubated with HRP-labeled goat anti-rabbit IgG (1:1000 dilution) for 2 hours at room temperature followed by three washes with TBST buffer.
(5) And finally, developing by using a developing solution and photographing for analysis.
4.4RT-PCR detection of the amount of EV71 Virus in cells
And (3) continuously culturing the Caco-2 cells for 48h after the Caco-2 cells are infected with the virus, extracting total RNA of the cells of the control group and the interference group by using TRIzol, carrying out reverse transcription to obtain cDNA, and detecting the amount of the EV71 virus by RT-PCR. The specific steps are as shown in 2.2.
The experimental results are as follows:
1 design, Synthesis and screening of effective siRNA
Aiming at each target gene sequence, the invention designs a plurality of RNA interference target sequences, and utilizes design software to perform pre-evaluation determination, 3 optimal kinetic parameter targets are selected to enter the subsequent experimental process, and each gene synthesizes 3 interference sequences in total, as shown in Table 1.
The method comprises the steps of transfecting the interfering RNA of each gene into Caco-2 cells by adopting an in vitro transfection method, detecting the relative expression quantity of each target gene (shown in table 2) by an RT-PCR method after 48 hours, and finally screening the siRNA sequence (the bold sequence in table 2) with the best interference effect for subsequent experiments.
TABLE 2RT-PCR method for detecting the relative expression of target genes after siRNA transfection
Note: CTRL: caco-2 cell group without any siRNA transfection (free-run control group);
NT: a Caco-2 cell group (negative control group) transfected with non-targeting siRNA;
siRNA: caco-2 cell group (experimental group) transfected with siRNA against each gene of interest.
2 interference efficiency and cytotoxicity assays after siRNA interference
The selected effective siRNA aiming at each host molecule is used for transfecting Caco-2 cells, the relative expression quantity of each target gene is detected by an RT-PCR method 48h after transfection, and the CCK8 is used for detecting the influence on Caco-2 cytotoxicity after transfection.
As shown in FIG. 1, the group transfected with effective siRNA was able to significantly suppress the expression level of the corresponding gene (P < 0.05) after transfection of each siRNA, compared with the group CTRL. The interference efficiency of the transfection FGFR1siRNA (SEQ ID NO:10) and IL4R siRNA (SEQ ID NO:21) is up to 83.36% and 70.59%, respectively.
The cytotoxicity experiment shows that after each siRNA transfection, obvious cytotoxicity (P is more than 0.05) is not generated, the normal physiological function of cells is not influenced, and the siRNA can be used for subsequent experiments.
3 Effect of siRNA interference on EV71 Virus infection
After the effective siRNA of each host molecule is transfected to down-regulate the expression of related molecules of host cells, EV71 virus with the same dosage is infected, after 48h of infection, the influence of the down-regulated host molecules on EV71 infection is detected by an immunofluorescence method, and the fact that the infection of Caco-2 cells by EV71 is obviously reduced after FGFR1 gene and IL4R gene are down-regulated by transfection of FGFR1siRNA (SEQ ID NO:10) and IL4R siRNA (SEQ ID NO:21) compared with a control group is found (FIG. 2A). Through calculation of virus amount, the inhibition rates of FGFR1 and IL4R genes on viruses after being down-regulated reach 87.90 percent and 90.58 percent respectively, while the down-regulation of the rest molecules does not obviously inhibit the infection of EV71 on Caco-2 cells (P > 0.05) (figure 2B).
To further clarify the important roles of FGFR1 and IL4R in EV71 infection, the effect on viral infectivity was observed after transfection of three sirnas against FGFR1 and IL4R molecules, respectively. The siRNA interference efficiency is detected by an RT-PCR method. The virus load of the EV71 is detected by an RT-PCR method and an immunoblotting method respectively. The results showed that different siRNAs interfered with different FGFR1 and IL4R molecules with different efficiencies (FIGS. 3A, 3B), with the FGFR1siRNA (SEQ ID NO:10) and IL4R siRNA (SEQ ID NO:21) interfering with the highest efficiency, consistent with the previous results. The influence of the siRNA of three FGFR1 molecules and three IL4R molecules on EV71 infectivity is detected, the inhibition rate of the siRNA to virus infection can reach more than 50%, and the virus amount in Caco-2 cells is also obviously reduced along with the increase of the interference efficiency to FGFR1 molecules and IL4R molecules (figures 4A-4D), which is consistent with the detection result of an immunofluorescence method. These results indicate that FGFR1 and IL4R molecules play an important role in EV71 infection of Caco-2 cells.
Therefore, FGFR1 and IL4R can be used as new host targets for inhibiting EV71 infection on Caco-2 cells.
The above experimental results prove that: the invention screens two new host cell molecules FGFR1 and IL4R which can inhibit EV71 from infecting Caco-2 cells. After the FGFR1 and IL4R genes are down-regulated, the normal physiological function of cells is not influenced, but the infection of the EV71 to Caco-2 cells is obviously inhibited. Therefore, the invention provides a new target and a treatment scheme for clinically preventing and treating EV71 infection.
While the preferred embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that the invention is not limited thereto, and that various changes and modifications may be made without departing from the spirit of the invention, and the scope of the appended claims is to be accorded the full scope of the invention.
Sequence listing
<110> second military medical university of China people liberation army
Application of fibroblast growth factor receptor 1in preparation of medicine for preventing and treating enterovirus 71 type infection
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Claims (3)
1. The application of the reagent for inhibiting or down-regulating the expression level of the fibroblast growth factor receptor 1in preparing the medicine for preventing or treating the enterovirus 71 infection; the reagent for inhibiting or reducing the expression quantity of the fibroblast growth factor receptor 1 refers to siRNA, shRNA, miRNA or antisense nucleotide which specifically interferes with the gene expression and processing of the fibroblast growth factor receptor 1, or a recombinant vector containing the siRNA, shRNA, miRNA or antisense nucleotide.
2. The use according to claim 1, wherein the use of the agent for inhibiting or down-regulating the expression level of fibroblast growth factor receptor 1in the preparation of a medicament for preventing or treating hand-foot-and-mouth disease, meningitis, brainstem encephalitis or poliomyelitis caused by enterovirus type 71 infection.
3. The use according to claim 1, wherein the agent that inhibits or down-regulates the expression level of fibroblast growth factor receptor 1is interfering RNA of fibroblast growth factor receptor 1, and the nucleotide sequence of the interfering RNA is shown in any one of SEQ ID NO. 10, SEQ ID NO. 11 and SEQ ID NO. 12.
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| CN106214701A (en) * | 2010-08-12 | 2016-12-14 | 菲特治疗公司 | The hematopoietic stem cell improved and progenitor cell therapy |
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| Title |
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| 瑞戈非尼的毒性反应管理现状;高杰，等;《现代医药卫生》;20170531;第33卷(第10期);1484-1487 * |
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