CN109568565B - Application of NF90 in preparing biological preparation for regulating and controlling osteogenic differentiation of bone marrow mesenchymal stem cells - Google Patents
Application of NF90 in preparing biological preparation for regulating and controlling osteogenic differentiation of bone marrow mesenchymal stem cells Download PDFInfo
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Abstract
The invention discloses application of NF90 in preparing a biological preparation for regulating and controlling osteogenic differentiation of bone marrow mesenchymal stem cells (BM-MSC). We found that RNA binding protein NF90 is essential for osteogenic differentiation of BM-MSCs, lncissi enhances mRNA stability of transcription factor HOXD8 by binding to NF90 protein in bone marrow mesenchymal stem cells (BM-MSCs) to promote osteogenic differentiation of BM-MSCs; therefore, NF90 can be used for preparing biological preparations for regulating and controlling the osteogenic differentiation of BM-MSC, such as plasmids, recombinant expression vectors, transgenic cell lines and genetically engineered bacteria which can over-express NF90 gene or can express shNF 90. The invention also discloses a pharmaceutical composition for preventing and/or treating Adolescent Idiopathic Scoliosis (AIS), which comprises an effective amount of NF90 and a pharmaceutically acceptable carrier, and is used for treating AIS associated with BM-MSC osteogenic differentiation abnormality in patients.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to application of RNA binding protein NF90 in preparing a biological preparation for regulating and controlling osteogenic differentiation of bone marrow mesenchymal stem cells (BM-MSC).
Background
Adolescent Idiopathic Scoliosis (AIS) is a complex three-dimensional deformity of the spine, occurring primarily in girls 10 to 16 years of age during Adolescent growth. The scoliosis may have deformity in appearance, back pain and dysfunction, and severe patients will have physiological function problems such as limited cardiopulmonary function and the like, and the social activities are influenced to different degrees. Epidemiological survey results show a global incidence of AIS in adolescents of about 2-4%, of which about 10% of people diagnosed with AIS require treatment. Current treatments for AIS include brace therapy and surgical correction; among them, full-time bracing therapy may cause back pain and psychological disorders, while corrective surgery using pedicle screw instruments inevitably results in severe surgical trauma, and even permanent catastrophic nerve or blood vessel injury when the screws are poorly positioned. If the risk of the onset and progression of AIS can be detected as early as possible, appropriate treatment regimens may be timely taken, reducing the pain and inconvenience associated with treatment delays. New advances in the etiology and pathogenesis of AIS, such as the exploration of the molecular mechanisms of AIS pathogenesis, will likely provide more convenient methods for AIS detection and for progression prediction and treatment.
Studies have shown that AIS patients have abnormal bone growth and a sustained reduction in Bone Mineral Density (BMD) compared to gender and age matched controls. In 1982 Burner et al first reported a reduction in bone mass in patients with AIS. Approximately one third of AIS patients have poor BMD. Low BMD is reported to be a key prognostic factor for AIS girl curve progression. It is believed that reduced bone mass may be a major factor in AIS spinal deformity. Many studies have demonstrated a 27% to 38% reduction in BMD in AIS patients. In addition, longitudinal follow-up on skeletal maturation showed that there was a continuous decrease in bone mass in more than 80% of AIS girls, suggesting that the decrease in bone mass may be a lifelong systemic abnormality in bone metabolism in AIS patients.
Mesenchymal Stem Cells (MSCs) are present in the stroma of all mammalian organs and can differentiate into osteoblasts, adipocytes and chondrocytes. In addition, MSCs are essential in intramembranous and endochondral bone formation. We have previously demonstrated that MSCs from Bone Marrow (BM) from AIS patients exhibit reduced osteogenic differentiation capacity. The study of Park et al further confirmed the results of our study. Our and other studies have shown that AIS patients develop abnormal differentiation of MSCs during development of osteoblasts, chondrocytes and adipocytes, and in view of the functional characteristics of Mesenchymal Stem Cells (MSCs) in bone formation and resorption, we speculate that the abnormal osteogenic differentiation of MSCs is associated with the pathogenesis of AIS. However, how MSCs are abnormally regulated in AIS patients remains elusive.
Non-coding RNA (ncRNA) is called 'dark matter' in a living body, and the proportion of ncRNA in the whole genome is closely related to the complexity level among biological species. The complex and precise regulation function of the ncRNA in development and gene expression explains the complexity of the genome, and opens a new way for people to know the complexity of a living body from the dimension of a gene expression regulation network. There is increasing evidence that the development of a range of major diseases is associated with an imbalance in the regulation of non-coding RNAs.
Long non-coding rnas (lncrnas) have recently been considered to be transcripts of more than 200 nucleotides (nt) of a class of genes that have no protein coding ability. lncRNA is less conserved in various species, but is more tissue-specific than protein-encoding genes. Research has shown that lncRNA plays a broad role in gene regulation and other cellular processes. lncRNA is involved in a variety of biological processes including chromatin modification, transcriptional regulation, imprinting, and nuclear transport. LncRNA performs its function through a variety of mechanisms, including co-transcriptional regulation, regulation of gene expression, scaffolding of nuclear or cytoplasmic complexes, and pairing with other RNAs. We have recently reported that several lncrnas are involved in self-renewal maintenance of liver cancer stem cells. Recent studies have reported that several lncrnas regulate their respective adjacent protein-encoding genes, playing a key role in mesendoderm differentiation and cardiac development. However, the biological role of lncRNA in AIS pathogenesis is not clear.
The NF90 protein is one of the most main protein isomers of the interleukin enhancer binding factor 3(ILF3) protein family, and the family protein interacts with coding and non-coding RNA in cells and virus initial ends and participates in multiple cell functions such as cell development, cell cycle and virus infection. Of these, NF90 is mainly present in the nucleus of the cell and is an RNA-binding protein that regulates gene expression or stabilizes mRNA.
The invention tries to explore the interaction relation between the lncRNA (i.e. lncAIS) related to AIS and NF90 protein in Mesenchymal Stem Cells (MSC) and the correlation between the lncRNA and the NF90 protein and the pathogenesis of AIS, thereby providing a more convenient method for treating AIS.
Disclosure of Invention
The invention aims to provide application of NF90 in preparing a biological preparation for regulating and controlling osteogenic differentiation of bone marrow mesenchymal stem cells (BM-MSC). We found that lncAIS enhances mRNA stability of transcription factor HOXD8 in BM-MSC by binding to NF90 protein to promote osteogenic differentiation of BM-MSC, NF90 being necessary for osteogenic differentiation of BM-MSC; therefore, NF90 can be used for preparing biological preparations for regulating and controlling osteogenic differentiation of bone marrow mesenchymal stem cells (BM-MSC), such as plasmids, recombinant expression vectors, transgenic cell lines and genetically engineered bacteria which can over-express NF90 gene or can express shNF 90.
The inventor of the present invention found in the related research on Adolescent Idiopathic Scoliosis (AIS) that LncAIS (gene symbol: ENST00000453347) is lncRNA significantly differentially expressed in bone marrow mesenchymal stem cells (BM-MSC) of AIS patients, which is significantly down-regulated in BM-MSC of AIS patients. The experiment of the invention proves that NF90 binds lncAIS in BM-MSC, and the lncAIS interacts with NF 90; we found that of the first 10 down-regulated transcription factors in selected lncas-silenced BM-MSCs, NF90 specifically binds to mRNA of the transcription factor HOXD8, and that mRNA of HOXD8 interacts with NF90 in BM-MSCs; the present invention demonstrates that lncAIS interacts with NF90 to maintain the stability of HOXD8 mRNA in normal BM-MSC, thereby promoting osteogenic differentiation of BM-MSC.
In some embodiments of the above application of the present invention, the biological agent for regulating osteogenic differentiation of BM-MSC comprises plasmid, recombinant expression vector, transgenic cell line, genetically engineered bacterium, which over-expresses NF90 gene or can express shNF 90. Further, in some embodiments of the invention, the plasmid overexpressing NF90 gene is pSIN-EF2 or the plasmid capable of expressing shNF90 is a pSicoR plasmid. Further, in other embodiments of the present invention, the sequence of shNF90 is shown in SEQ ID NOs.7-9.
The invention also aims to provide a biological preparation for regulating and controlling osteogenic differentiation of bone marrow mesenchymal stem cells, which comprises a plasmid, a recombinant expression vector, a transgenic cell line and a genetically engineered bacterium, wherein the plasmid can over-express NF90 gene or can express shNF 90. In some embodiments of the biological agent of the present invention, the plasmid overexpressing NF90 gene is pSIN-EF2 or the plasmid capable of expressing shNF90 is a pSicoR plasmid. Further, in other embodiments of the present invention, the sequence of shNF90 is shown in SEQ ID NOs.7-9.
Another aspect of the present invention provides a use of NF90 in the manufacture of a pharmaceutical composition for the prevention and/or treatment of Adolescent Idiopathic Scoliosis (AIS), wherein the pharmaceutical composition comprises an effective amount of NF90 and a pharmaceutically acceptable carrier.
Yet another aspect of the present invention provides a pharmaceutical composition for the prevention and/or treatment of Adolescent Idiopathic Scoliosis (AIS) in a patient, characterized in that said pharmaceutical composition comprises an effective amount of NF90 and a pharmaceutically acceptable carrier, optionally said pharmaceutical composition further comprises a further agent for the prevention or treatment of AIS.
As used herein, the term "down-regulation" or "down-regulation" refers to, for example, for a specific nucleotide sequence, such as a specific lncRNA sequence or NF90 gene sequence, a measurement of the amount of the sequence indicating a reduced level of expression of the sequence, for example, in a biological sample isolated from an AIS patient or an individual at risk of AIS, such as BM-MSC, compared to a normal individual. Conversely, "expression up-regulation" or "up-regulation" means that, for example, for a specific nucleotide sequence, such as a specific lncRNA sequence or NF90 gene sequence, the measurement of the amount of the sequence indicates an increased level of expression of this sequence, for example, in a biological sample isolated from an AIS patient or an individual at risk of AIS, such as BM-MSC, compared to a normal individual.
As used herein, the term "individual", "subject" or "patient" includes, but is not limited to, humans and other primates (e.g., chimpanzees and other apes and monkey species). In some embodiments, the subject or patient is a human.
In the present invention, the term "lncAIS (Ensembl genome database gene symbol: ENST 00000453347)" refers to lncRNA having an original sequence shown by the gene in GeneBank, which is currently the international common nucleic acid database, and includes lncRNA of natural or synthetic origin. IncAIS analogue refers to a derivative or variant form of the IncRNA which is substituted, deleted or added with one or more nucleotides, or is biologically modified and still has biological activity.
In the present invention, the term "HOXD 8(NCBI database gene ID: 3234)" refers to HOXD8 having the original sequence shown by HOXD8 gene in GeneBank, the present international common nucleic acid database, which includes HOXD8 and its analogs, which are naturally or synthetically derived. HOXD8 analog refers to a derivative or variant form thereof, which is substituted, deleted or added with one or several nucleotides, or which is biologically modified and still biologically active.
In the present invention, the term "NF 90(NCBI database gene ID: 3609)" refers to NF90 having the original sequence shown in ILF3 gene in GeneBank, which is the current International consensus nucleic acid database, and includes NF90 and its analogs, whether of natural or synthetic origin. NF90 analogs refer to derivatives or variant forms thereof that have been substituted, deleted or added with one or more nucleotides, or that have been biologically modified to remain biologically active.
The effective dosage of NF90 in the present invention may be adjusted accordingly depending on the mode of administration and the severity of the disease to be treated, etc. The preferred effective amount can be determined by one of ordinary skill in the art by combining various factors. Such factors include, but are not limited to: pharmacokinetic parameters of NF90, health of the treated patient, body weight, route of administration, and the like.
In some embodiments of the invention, the vector to which NF90 is bound may be a vector of the type commonly used in the art for expression of NF90 in host cells, such as a liposome, chitosan, or lentiviral expression vector, and the pharmaceutically acceptable excipients include various excipients, diluents, and adjuvants that are used in medicine without causing significant side effects, including but not limited to: purified water, physiological saline, buffer, glucose, water, glycerol, mannitol, ethanol, surfactants and salts such as sodium chloride, sodium EDTA and the like.
The pharmaceutical compositions of the present invention may include classical pharmaceutical formulations. The pharmaceutical composition according to the present invention may be administered by any conventional route as long as the target tissue is available by the route.
In some embodiments of the invention, the expression vector is a lentiviral expression vector, preferably the lentiviral expression vector may be a pSicoR, pSIN-EF2 or pWPXL plasmid, wherein the NF90, preferably pSIN-EF2, plasmid is overexpressed and the NF90, preferably pSicoR, plasmid is knocked down.
In some embodiments of the invention, the pharmaceutical composition further optionally comprises one or more other agents effective in treating AIS, which agents are well known to those skilled in the art. The pharmaceutical composition of the present invention may be administered in combination with other therapeutic means for the prevention and/or treatment of AIS.
Has the advantages that:
the inventors of the present invention found that RNA binding protein NF90 is associated with osteogenic differentiation, lnciss enhances mRNA stability of transcription factor HOXD8 by binding to NF90 protein in bone marrow mesenchymal stem cells (BM-MSCs) to promote osteogenic differentiation of BM-MSCs, NF90 is necessary for osteogenic differentiation of BM-MSCs; therefore, NF90 can be used for preparing biological preparations for regulating and controlling osteogenic differentiation of bone marrow mesenchymal stem cells (BM-MSC), such as plasmids, recombinant expression vectors, transgenic cell lines and genetically engineered bacteria which can over-express NF90 gene or can express shNF 90. The invention also discloses a pharmaceutical composition for preventing and/or treating Adolescent Idiopathic Scoliosis (AIS), which comprises an effective amount of NF90 and a pharmaceutically acceptable carrier, and is used for treating AIS associated with BM-MSC osteogenic differentiation abnormality in patients.
Drawings
The results shown in fig. 1a to h demonstrate that LncAIS are down-regulated in the BM-MSCs of AIS patients. In particular, the amount of the solvent to be used,
FIG. 1a is a graph showing the cluster analysis of differentially expressed lncRNA obtained by microarray analysis of BM-MSC of healthy donors and AIS patients, where lncAIS is the lncRNA with the greatest downregulation in BM-MSC of AIS patients.
Figure 1b shows that LncAIS is located on human chromosome 1, which is identified as a conserved locus.
FIG. 1c shows comparison of LncAIS transcripts in Normal healthy donors (normals) and AIS patient BM-MSCs by real-time qPCR, indicating significant downregulation of lncAIS in AIS patient-derived BM-MSCs.
FIG. 1d shows comparison of LncAIS expression in BM-MSC of Normal healthy donors (Northern) and AIS patients by Northern blotting, indicating that lncAIS is down-regulated in BM-MSC of AIS patients.
FIG. 1e shows that LncAIS has no coding potential as analyzed by the coding Capacity assessment tool (CPAT), where XIST transcript was used as the non-coding gene control and GAPDH and RUNX2 were used as the coding gene control.
FIG. 1f shows the results of the outer translation assay using pcDNA4-myc-his plasmid, which KLF4 was used as a protein-encoding control, showing that lncAIS does not produce any detectable peptide.
The BM-MSC cell fractionation assay results shown in FIG. 1g indicate that lncAIS is distributed mainly in the nuclei of human BM-MSC with HMBS RNA, ACTIN RNA and GAPDH RNA as positive controls for cytoplasmic gene expression and U1RNA as a positive control for nuclear gene expression.
The results of the RNA fluorescent in situ hybridization (RNA-FISH) assay shown in FIG. 1h indicate that lncAIS is down-regulated in the BM-MSC of AIS patients relative to Normal healthy donors (Normal), with red: lncAIS probe (probe); green: actin (Actin); nuclei were counterstained with DAPI.
The results shown in a to l in fig. 2 demonstrate that LncAIS interact with NF90 to enhance mRNA stability of HOXD 8. In particular, the amount of the solvent to be used,
figure 2a shows that the consumption of lnciss (shL ncAIS) in normal BM-MSCs by RT-qPCR assessment did not affect the expression level of its neighboring genes, where NS indicates no significance, indicating that lnciss may exert its regulatory role in trans.
Figure 2b shows a biotin-labeled RNA knock-down assay to identify possible lncas-related proteins from BM-MSC lysates, indicating that NF90 binds lncas in BM-MSC.
Figure 2c shows that lncas interaction with NF90 was verified by RNA pull-down (knock-down) assay followed by immunoblot analysis by anti-NF 90 antibody.
FIG. 2d shows incubation of BM-MSC lysates with Anti-NF 90(Anti-ILF3) antibody, verifying the interaction of lncAIS with NF90 by immunoprecipitation (RIP) assay.
FIG. 2e shows the co-localization of lncAIS with NF90 in the nucleus of BM-MSC. BM-MSCs were probed with lncAIS by RNA-FISH, followed by immunofluorescent staining for NF 90. Red: a lncAIS probe; green: NF 90; nuclei were counterstained with DAPI.
FIGS. 2F and 2g show that infection of normal BM-MSCs with lentiviruses expressing shNF90, cultured in MSC medium (FIG. 2F) and OriCell MSC osteogenic differentiation medium (FIG. 2g), respectively, revealed that NF90 depletion (shNF90) suppressed the expression levels of self-renewal-associated genes (NANOG, POU5F1, and SOX2) (FIG. 2F) and osteogenic differentiation genes (ALPL, BSP, RUNX2, LPL, and PPAR) (FIG. 2g) in BM-MSCs, as assessed by real-time qPCR.
FIGS. 2h and 2i show that ALP staining (FIG. 2h) and Von Kossa staining (FIG. 2i) of normal BM-MSCs cultured in OriCell MSC osteogenic differentiation medium after infection with lentivirus expressing shNF90 indicate that NF90 depletion (shNF90) inhibits osteogenic differentiation of BM-MSCs.
FIG. 2j shows analysis of the interaction of mRNA designated in BM-MSC with NF90 by RIP assay; incubation of BM-MSC lysates with anti-NF 90 antibody followed by RNA immunoprecipitation and real-time qPCR revealed that NF90 specifically binds to mRNA of HOXD8 in the first 10 downregulated transcription factors in selected lncas-silenced BM-MSCs.
FIG. 2k shows validation of the interaction of mRNA of HOXD8 with NF90 in BM-MSC by RIP assay; BM-MSC lysates were incubated with anti-NF 90 antibody and then subjected to RIP assay. Also as shown in figure 2l, HOXD8 mRNA stability from the indicated BM-MSCs was measured by real-time qPCR at the indicated time points after Act D treatment. The results show that lnciss depletion (shLncAIS) in BM-MSCs abolished the interaction of NF90 with the 3' -UTR region of HOXD8 mRNA (fig. 2k) and thus resulted in attenuation of HOXD8 mRNA (fig. 21). Consistently, the interaction of NF90 with the 3' -UTR region of HOXD8 mRNA in the BM-MSCs of AIS patients was undetectable (fig. 2k), and also abrogated the stability of HOXD8 mRNA (fig. 2 l).
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.
The following are materials and general methods used in the examples of the present invention
Antibodies and reagents
Antibodies to human HOXD8(ab228450) and NF90(ab89100) were purchased from Abcam (Cambridge, USA). Anti- β -actin (clone AC-74) antibody was from Sigma-Aldrich (st. louis, USA). Antibodies against Myc (clone 9E10) were from Santa Cruz Biotechnology (Santa Cruz, USA). Antibodies against COL1A1(BA0325), IBSP (BA2329) and OPN (PA1432) were from Boster (Wuhan, China). Secondary antibodies conjugated to Alexa-594 were purchased from Molecular probes Inc (Eugene, USA). Streptavidin beads were from Sigma-Aldrich (st. louis, USA). Protein A/G beads were from Santa Cruz Biotechnology (Santa Cruz, USA). Alkaline phosphatase detection kits were purchased from Millipore (Millerica, USA). SuperReal premix plus qPCR buffer was from TIANGEN Biotech (Beijing, China). The oriCell BM-MSC osteogenic differentiation kit is from Cyagen (HUXMA-90021, China). The Von Kossa staining kit is from Genmed sciences (shanghai, china). Cell counting kit-8 (CCK-8) was from Dojindo (Kumamoto, Japan). The Fast Green staining kit and the Alcian Blue staining kit were from Xinhuaalvuan Biotechnology (Beijing, China).
The probe, the primer, the shLncAIS and shNF90 gene sequences of the invention
Table 1: lncAIS (11-228nt) probe sequence for RNA FISH
| lncAIS probe | Probe sequence | Sequence numbering |
| |
5’-CTTTCCCTGAGAAAAACCTCC-3’ | SEQ ID NO:1 |
| lncAIS Probe #2 | 5’-AGGATTAGGAAGCCTCCTGC-3’ | SEQ ID NO:2 |
| lncAIS Probe #3 | 5’-CGCTTCTCTCTTCTACTGTCC-3’ | SEQ ID NO:3 |
Table 2: shLncAI gene sequence for lncAIS knock-down
| Gene | Gene sequences | Sequence numbering |
| |
5’-TTCCTAATCCTGCTCCAG-3’ | SEQ ID NO:4 |
| shLncAIS#2 | 5’-ATAGACATCTGGTTTCTGG-3’ | SEQ ID NO:5 |
| shLncAIS#3 | 5’-TTAGGACATAGACATCTG-3’ | SEQ ID NO:6 |
Table 3: shNF90 gene sequence for NF90 knock-down
| Gene | Gene sequences | Sequence numbering |
| |
5’-AAACCCAGTGAAGCACAGG-3’ | SEQ ID NO:7 |
| shNF90#2 | 5’-AAGCCTGTCTGTTTCTTGC-3’ | SEQ ID NO:8 |
| shNF90#3 | 5’-AATCCCATGCATCTGCAGC-3’ | SEQ ID NO:9 |
Table 4: primer sequences for cDNA amplification in qRT-PCR analysis
Patient and sample
Bone Marrow (BM) aspirates were obtained from 42 AIS patients (mean age 14.5 years, range 12-17 years) and 25 healthy donors (mean age 14.9 years, range 12-17 years). In the AIS group, all patients received a comprehensive clinical and radiological examination to exclude other causes of scoliosis and to determine a diagnosis of AIS. In the control group, each of 25 age and gender matched subjects had a straight spine and normal forward curvature test by physical examination. Upon entry into the study, they were confirmed to be free of any associated medical illness or spinal deformity. The study was approved by the ethical committee of the Chinese academy of medical sciences and the Beijing-coordinated hospital. Written informed consent was obtained from all subjects and their parents prior to study entry.
Cell isolation, culture and osteogenic differentiation assay
Human bone marrow tissue was collected from AIS patients and healthy donors. All experiments were performed according to the procedure approved by the ethical committee of the chinese medical academy of sciences and the cooperation of beijing with hospitals. Human BM-MSCs were isolated and cultured as described below (Zhuang Q, Mao W, Xu P, Li H, Sun Z, Li S, et al. identification of Differential Genes Expression Profiles and Pathways of Bone Marrow genetic Stem Cells of additive Idiopathic stresses tissue by micro and Integrated Gene Network analysis. spine 2016,41(10):840 855). Human 293T cells were cultured in DMEM supplemented with 10% FBS and 100U/ml penicillin and 100mg/ml streptomycin. Lentiviruses were produced in 293T cells using standard protocols. Transfection was performed using lipofectin (Invitrogen). For shRNA knockdown and overexpression experiments, the target sequence was constructed into the pSicoR plasmid. Lentiviruses are produced by 293T cells. The most potent shRNA among the 3 shRNA constructs was selected for the following experiment. Biological replicates were performed using three independent knockdown cell lines in each assay. At least four independent experiments were performed as biological replicates. For example, the sequence of the shlncAIS gene used for the knockdown of lncAIS is shown as SEQ ID NOS: 4-6 in Table 2 above; the shNF90 gene sequence for NF90 knock-down is shown as SEQ ID NOS: 7-9 in Table 3 above. To induce osteogenic differentiation, third generation BM-MSCs were seeded in six-well plates and treated with osteogenic induction medium according to the manufacturer's protocol. The medium was changed every 3 days.
Wound healing test
The dishes were coated with 0.1% gelatin (v/v) for 1 hour at 37 ℃. Will be 1 × 106Individual BM-MSC cells were plated to generate confluent monolayers. Culturing cellsTo fully adhere and diffuse. Wounds were created by manually scraping cell monolayers with a p200 pipette tip. A first image is acquired using the reference point markers. Cells were cultured in a tissue culture incubator for 24 hours. The second image is acquired by matching the shooting area of the first image.
Coding potential analysis
The coding potential of lncAIS was analyzed on the website of http:// lilab. research.bcm.edu/CPAT/by the Coding Potential Assessment Tool (CPAT) according to the manufacturer's instructions. XIST transcript was used as a non-coding gene control. GAPDH and RUNX2 were used as coding gene controls.
Cell fractionation analysis
BM-MSCs were lysed using NE-PER nuclear and cytoplasmic extraction kit (Pierce) according to the manufacturer's instructions, followed by nuclear and cytoplasmic fractionation. RNA was extracted using TRIzol Reagent (Invitrogen) and then purified using RNeasy kit (Qiagen, Valencia, Calif., USA). Reverse transcription was performed by M-MLV reverse transcriptase (Promega) and qRT-PCR analysis. ACTIN RNA and GAPDH RNA were used as positive controls for cytoplasmic gene expression. U1RNA served as a positive control for nuclear gene expression.
ALP and Von Kossa staining
ALP staining was monitored using the ALP staining kit according to the manufacturer's protocol. Mineral deposition was monitored using Von Kossa staining kit according to the manufacturer's protocol. Images were obtained with a Nikon EclipseTi microscope (Nikon, Japan). The colour intensity of mineral deposits was quantified by ImageJ.
Northern blotting
Total RNA was extracted from BM-MSC using TRIzol. Mu.g of RNA from each sample was subjected to formaldehyde denaturing agarose electrophoresis and then transferred to positively charged NC membranes using 20 XSSC buffer (3.0M NaCl and 0.3M sodium citrate, pH 7.0). The membrane is UV cross-linked and incubated with a biotin-labeled RNA probe, such as the IncAIS (11-228nt) probe, produced by in vitro transcription. Detection of biotin signal with HRP-conjugated streptavidin was used in the cheniuuminescent nucleic acid detection module according to the manufacturer's instructions.
RNA FISH
Fluorescently conjugated lncas probes were generated according to the protocol of Biosearch Technologies. The sequences of the lncAIS (11-228nt) probe set for RNA FISH are shown in SEQ ID NOS: 1-3 in Table 1 above. BM-MSCs were hybridized to DNA probe sets and then stained with the indicated antibodies. Images were obtained with an Olympus FV1200 laser scanning confocal microscope (Olympus, Japan).
Microarray analysis
RNA was extracted from BM MSCs using TRIzol Reagent (Invitrogen) and then purified using RNeasy kit (Qiagen, Valencia, CA, USA) according to the manufacturer's instructions. cDNA was generated using One-Cycle Target Labeling and Control Reagents (Affymetrix, Santa Clara, Calif., USA) and cRNA was generated using the GeneChip WT Labeling kit (Affymetrix, Santa Clara, Calif., USA). Biotin-labeled fragmented (. ltoreq.200 nt) cRNA was hybridized with Affymetrix GeneChip Human transcript array 2.0(Affymetrix) at 45 ℃ for 16 h. GeneChip was washed and stained in Affymetrix Fluidics Station 450. Use and install in
Scanner 30007G
GeneChipCommand Console (AGCC) scans GeneChips. Data were analyzed using the Robust Multichip Analysis (RMA) algorithm using Affymetrix default Analysis settings and global scaling as normalization methods. The values given are log2 RMA signal strength. Microarray data was stored in GEO under accession number (GSE 110359).
RNA knock-down assay
Biotin-labeled lncas full length (sense) and antisense RNA were obtained in vitro with biotin RNA labeling cocktail (Roche) and then incubated with extracts isolated from BM-MSCs. The RNA binding protein was pulled down (pull down) by streptavidin beads. The pull-down fractions were separated by SDS-PAGE and then silver stained. Differential bands enriched by lncas were analyzed by LTQ Orbitrap XL mass spectrometry or immunoblotted with the indicated antibodies.
RNA Immunoprecipitation (RIP) assay
BM-MSCs were treated with 1% formaldehyde and then solubilized with RNase-free RIPA buffer (50mM Tris-HCl [ pH 7.4], 150mM NaCl, 0.5% sodium deoxycholate, 0.1% SDS, 5mM EDTA, 2mM PMSF, 20mg/ml aprotinin, 20mg/ml leupeptin, 10mg/ml pepstatin A, 150mM benzamidine, 1% Nonidet P-40, and RNase inhibitor). Samples were sonicated on ice and centrifuged. The supernatant was previously cleared and incubated with the indicated antibodies, followed by protein a/G bead immunoprecipitation. Extracting total RNA from the eluate. LncAIS enrichment was analyzed by qPCR. The primer sequences for cDNA amplification are listed in Table 4.
Chromatin immunoprecipitation (ChIP) assay
ChIP was quantified according to standard protocol (Upstate). Will be selected from BM-MSC (2X 10) fixed in 1% formaldehyde6) Sheared chromatin (sonicated to 200-500bp) was incubated with 4 μ g of antibody overnight at 4 ℃ and then immunoprecipitated with salmon sperm DNA/protein agarose beads. After washing, elution, and cross-linking reversal, DNA from each ChIP sample and the corresponding input sample was purified and analyzed using qPCR. The primer sequences for cDNA amplification are listed in Table 4.
Ectopic bone formation in vivo
Will be 2X 10 in total6The individual BM-MSC were incubated with approximately 100mg of wet HA/TCP ceramic powder (National Engineering Research Center for Biomaterials, Chengdu, China) overnight at 37 ℃. Cells were implanted subcutaneously on the dorsal surface of 8-week-old NOD/SCID mice. The implants were harvested after 8 weeks, fixed in 4% paraformaldehyde, decalcified in 10% EDTA, embedded in paraffin, then sectioned and stained. The bone tissue was stained green by rapid green staining. Cartilage tissue was stained blue by alcian blue staining to indicate bone maturation.
Statistical analysis
Unpaired student's t-test was used as a statistical analysis in the present invention. Statistical calculations were performed using Microsoft Excel or SPSS 13. When P <0.05, the P value was significant.
Examples
Example 1: identification of LncAS as a significantly differentially expressed lncRNA that is down-regulated in bone marrow mesenchymal stem cells (BM-MSC) of AIS patients
To identify key lncrnas involved in Adolescent Idiopathic Scoliosis (AIS), we performed microarray analysis of BM-MSCs from 5 healthy donors and 12 AIS patients. 1483 lncRNA showed differential expression in normal BM-MSC and AIS patients' BM-MSC, with 718 up-regulated and 765 down-regulated, as shown in FIG. 1 a. Among the lncRNAs with the greatest downregulation amplitude in BM-MSCs of AIS patients, we focused on the uncharacterized lncRNA which we called lncAIS (gene symbol: ENST 00000453347). lncas is located on human chromosome 1, contains 4 exons, and is a full-length 476nt transcript. lncas were identified as conserved loci as shown in figure 1 b.
Real-time qPCR was performed according to the conventional procedure to analyze IncAIS transcripts in BM-MSC of normal healthy donors and BM-MSC of AIS patients, and cDNA primers for amplification of IncAIS were shown as SEQ ID NO:10-11 in Table 4 above. BM-MSC were from 20 healthy donors and 30 AIS patients. Relative gene expression fold was normalized to endogenous β -actin and counted as mean ± s.d. P < 0.01. The results demonstrate a significant downregulation of lnciss in AIS patient derived BM-MSCs compared to BM-MSCs from healthy donors, as shown in figure 1 c.
IncAIS expression in BM-MSC of normal healthy donors and in BM-MSC of AIS patients was examined by Northern blot. The probes for lncAIS (11-228nt) of 217nt, whose sequences are shown in SEQ ID NOS: 1-3 in Table 1 above, were labeled and used for northern blot analysis. RNA was extracted from designated normal healthy donor BM-MSC and AIS patient BM-MSC, respectively, and 18S rRNA (1482-1725nt) was used as loading control. BM-MSC were from 3 healthy donors and 3 AIS patients. As shown in fig. 1d, Northern blot further confirmed lncas down-regulation in BM-MSC of AIS patients, showing only one transcript of lncas.
Analysis by a coding ability assessment tool (CPAT) showed that lncas has no coding potential; where XIST transcripts were used as non-coding gene controls and GAPDH and RUNX2 were used as coding gene controls, as shown in FIG. 1 e. The lncAIS transcript was cloned into pcDNA4-myc-his plasmid and transfected into 293T cells for 48 hours. Expression of Myc-fusion protein was analyzed by immunoblotting with anti-Myc antibody. KLF4 was used as a control for coding proteins. In vitro translation assays showed that lncas did not produce any detectable peptides, as shown in figure 1 f.
BM-MSC is firstly cracked, then nuclear and cytoplasmic fractionation and RNA extraction are carried out, and then qRT-PCR analysis is carried out. HMBS RNA, ACTIN RNA and GAPDH RNA were used as positive controls for cytoplasmic gene expression. The U1RNA served as a positive control for nuclear gene expression. N: the nuclear fraction. C: cytoplasmic fraction. The primer pairs used for cDNA amplification in the qRT-PCR analysis are listed in table 4 above. Data were from three independent experiments using BM-MSCs derived from 3 healthy donors. Cell fractionation assay results showed that lncAIS is mainly distributed in the nuclei of human BM-MSC, as shown in FIG. 1 g.
lncAIS was visualized in BM-MSC by RNA fluorescent in situ hybridization (RNA-FISH) assay followed by immunofluorescent staining. Red: a lncAIS probe; green: actin; nuclei were counterstained with DAPI. Scale bar, 20 μm. The lncAIS probe sequence is shown in SEQ ID NO 1-3 in Table 1 above. Over 100 representative cells were observed. RNA fluorescence in situ hybridization (RNA-FISH) further demonstrated the downregulation of lncAIS in BM-MSC of AIS patients and the distribution of lncAIS in nuclei, as shown in FIG. 1 h.
In conclusion, we revealed that lncAIS is highly expressed in normal human BM-MSC, but is significantly down-regulated in AIS patients BM-MSC.
Example 2: determination of LncAIS interaction with NF90 to enhance mRNA stability of HOXD8
LncRNA is normally positively associated with the regulation of its nearby protein-encoding gene. However, as shown in FIG. 2a, we found that the consumption of lncAIS in BM-MSC did not affect the expression level of its neighboring genes as assessed by qRT-PCR (the sequence of the shlncAIS gene for lncAIS knock-down is shown as SEQ ID NO:4-6 in Table 2 above), where the relative fold change in gene expression was calculated as mean. + -. S.D. NS, not significant. Data were from three independent experiments using BM-MSCs derived from 3 healthy donors. This suggests that lnciss may exert its regulatory role in trans.
A biotin-labelled RNA knock-down assay was then performed to identify possible lncas-related proteins from BM-MSC lysates. Using full length lncas transcript (sense) and antisense sequence controls, followed by mass spectrometry, biotin-RNA pull-down with lysates of BM-MSCs was performed, as shown in fig. 2b, to determine that NF90 binds lncas in BM-MSCs. NF90 is a protein of the interleukin enhancer binding factor 3(ILF3) family, an RNA binding protein that regulates gene expression or stabilizes mRNA.
As shown in fig. 2c, the interaction of lncas with NF90 was verified by RNA pull-down assay, followed by immunoblot analysis by anti-NF 90 antibody. The shNF90 gene sequence for NF90 knock-down is shown as SEQ ID NO 7-9 in Table 3 above; data were from three independent experiments using BM-MSCs derived from 3 healthy donors.
BM-MSC lysates were incubated with anti-NF 90 antibody and then subjected to RIP assay. RNA was extracted and reverse transcribed, LncAIS transcripts were analyzed by real-time qPCR, and the primer pairs used for cDNA amplification in qRT-PCR are listed in table 4 above. Data were from three independent experiments using BM-MSCs derived from 3 healthy donors. As shown in fig. 2d, the interaction of lncas with NF90 was verified by immunoprecipitation (RIP) assay.
The results of RNA knockdown (fig. 2c) and RNA-immunoprecipitation (RIP) assay (fig. 2d) confirm the interaction of lncas with NF 90.
As shown in FIG. 2e, lncAIS was co-localized with NF90 in the nucleus of BM-MSC. BM-MSCs were probed with lncAIS by RNA-FISH, followed by immunofluorescent staining for NF 90. Red: a lncAIS probe; green: NF 90; nuclei were counterstained with DAPI. Scale bar, 50 μm. The lncAIS probe sequence is shown in SEQ ID NO 1-3 in Table 1 above. Over 100 representative cells were observed. Data were from three independent experiments using BM-MSCs derived from 3 healthy donors.
The above experimental data indicate that lncAIS binds to NF90 protein in BM-MSC.
To further determine how NF90 regulates osteogenic differentiation and the pathogenesis of AIS, we depleted NF90 in normal BM-MSCs by lentiviral-mediated shRNA.
As shown in fig. 2f, BM-MSCs were infected with lentiviruses expressing shNF90 and cultured in MSC medium for 3 days, and the expression level of self-renewal-associated genes was evaluated by real-time qPCR. BM-MSCs were cultured in OriCell MSC osteogenic differentiation medium for 6 days, and the expression level of osteogenic differentiation genes was evaluated in the designated BM-MSCs by real-time qPCR, as shown in FIG. 2 g. The primer pairs used for cDNA amplification in qRT-PCR are listed in table 4 above. Relative gene expression fold changes were normalized to endogenous β -actin and counted as mean ± s.d. P < 0.01. Data were from three independent experiments using BM-MSCs derived from 3 healthy donors. The results shown in FIGS. 2F and 5g indicate that NF90 depletion suppressed the expression levels of self-renewal-associated genes (NANOG, POU5F1, and SOX2) and osteogenic differentiation genes (ALPL, BSP, RUNX2, LPL, and PPAR) in BM-MSC.
BM-MSCs were cultured in OriCell MSC osteogenic differentiation medium for 6 days to induce osteogenic differentiation, and ALP staining was performed on day 6 of osteogenic differentiation, as shown in fig. 2 h. BM-MSCs were cultured in OriCell MSC osteogenic differentiation medium for 6 days to induce osteogenic differentiation, and Von Kossa staining was performed to indicate mineral deposition on day 12, as shown in fig. 2 i. Data were from three independent experiments using BM-MSCs derived from 3 healthy donors. Scale bar, 10 μm. The colour intensity of mineral deposits was quantified by ImageJ and the intensity variation was calculated as mean ± s.d. P < 0.01. Results of ALP staining (fig. 2h) and Von Kossa staining (fig. 2i) demonstrate inhibited osteogenic differentiation.
The above experimental data indicate that NF90 is essential for osteogenic differentiation of BM-MSC.
NF90 is reported to interact with the 3' -UTR region of PARP1mRNA to maintain its mRNA stability. We analyzed the interaction of the designated mRNA in BM-MSC with NF90 by RIP assay. BM-MSC lysates were incubated with anti-NF 90 antibody, followed by RNA immunoprecipitation and real-time qPCR. Data were from three independent experiments using BM-MSCs derived from 3 healthy donors. As shown in fig. 2j, of the first 10 down-regulated transcription factors in lncas-silenced BM-MSCs we selected, NF90 specifically binds to mRNA of HOXD 8.
The interaction of mRNA from HOXD8 with NF90 in BM-MSC was verified by RIP assay. BM-MSC lysates were incubated with anti-NF 90 antibody and then subjected to RIP assay. Data were from three independent experiments using BM-MSCs from 3 healthy donors, as shown in figure 2 k. As shown in fig. 2l, HOXD8 mRNA stability from the indicated BM-MSCs was measured by real-time qPCR at the indicated time points after Act D treatment. Data are shown as mean ± s.d., P < 0.01. Data were from three independent experiments using BM-MSCs derived from 3 healthy donors.
The results shown in fig. 2k indicate that lnciss consumption in BM-MSCs abolished the interaction of NF90 with the 3' -UTR region of HOXD8 mRNA and thus resulted in attenuation of HOXD8 mRNA (fig. 21). Consistently, as shown in fig. 2k, the interaction of NF90 with the 3' -UTR region of HOXD8 mRNA in the BM-MSC of AIS patients was undetectable and also abrogated the stability of HOXD8 mRNA (fig. 2 l).
The above experimental results show that lnciss interacts with NF90 to maintain stability of HOXD8 mRNA in normal BM-MSCs.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Sequence listing
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Claims (5)
- The application of NF90 and lncAIS in preparing a biological preparation for regulating and controlling osteogenic differentiation of bone marrow mesenchymal stem cells, wherein the Ensembl genome database gene symbol of the lncAIS is ENST 00000453347.
- 2. The use of claim 1, wherein lnciss enhances mRNA stability of transcription factor HOXD8 by binding to NF90 protein in bone marrow mesenchymal stem cells to promote osteogenic differentiation of bone marrow mesenchymal stem cells.
- 3. The use of claim 1 or 2, wherein the biological agent for regulating osteogenic differentiation of bone marrow mesenchymal stem cells comprises a plasmid, a recombinant expression vector, a transgenic cell line, a genetically engineered bacterium, which overexpresses NF90 gene or can express shNF 90.
- 4. The use of claim 3, wherein said lentiviral expression vector capable of expressing shNF90 is a pSicoR plasmid.
- 5. The use of claim 3, wherein the shNF90 has the sequence shown in SEQ ID NOs 7-9.
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