CN115851608B

CN115851608B - Gene editing mesenchymal stem cell and preparation method and application thereof

Info

Publication number: CN115851608B
Application number: CN202211660570.4A
Authority: CN
Inventors: 曾彩芳; 林冠丽; 余蓓信
Original assignee: Second Affiliated Hospital of Guangzhou Medical University
Current assignee: Second Affiliated Hospital of Guangzhou Medical University
Priority date: 2022-12-23
Filing date: 2022-12-23
Publication date: 2023-08-01
Anticipated expiration: 2042-12-23
Also published as: CN115851608A

Abstract

The invention discloses a gene editing mesenchymal stem cell and a preparation method and application thereof, and relates to the technical field of biological medicine. The preparation method of the gene editing mesenchymal stem cells comprises the following steps: connecting a STAT3 promoter with the UBE3A fragment to construct a lentiviral expression vector; and transfecting the slow virus expression vector into the mesenchymal stem cells, and screening to obtain the gene editing mesenchymal stem cells. The gene-edited mesenchymal stem cells prepared by the invention highly express UBE3A, promote the ubiquitination of METTL1 and the degradation of METTL1 protein, can promote the differentiation efficiency of the mesenchymal stem cells to the cartilage forming direction, and provide a new treatment way for the repair of osteoarthritis cartilage.

Description

Gene editing mesenchymal stem cell and preparation method and application thereof

Technical Field

The invention relates to the technical field of biological medicines, in particular to a gene editing mesenchymal stem cell and a preparation method and application thereof.

Background

Osteoarthritis is also called degenerative osteoarthritis and osteoarthropathy, and mainly refers to degeneration and abrasion of articular cartilage and exposure of subchondral bone caused by aging of articular cartilage or increase of strain and activity and overmuch. Current methods of treating osteoarthritis are largely divided into non-surgical and surgical treatments. Non-surgical treatment is mainly pain management, and reduces joint pain and inflammatory response through application of non-steroidal anti-inflammatory drugs, intra-articular hyaluronic acid, glucocorticoids and the like. The most common method of surgical treatment is artificial joint replacement. The artificial joint replacement can relieve the symptoms of patients, effectively recover the joint function and improve the life quality of the patients. Non-surgical treatment only addresses pain and inflammation problems, but not others. Surgical treatment cannot solve the problem of joint integrity loss caused by cartilage damage. In the surgical treatment process, the surgical cost is high, and serious pressure is caused to families of patients and social medical resources; meanwhile, the artificial joint is not a body tissue, and can not be integrated with a self bone system, so that long-term complications such as loose and sinking of the prosthesis can be avoided, the operation is failed, secondary revision operation is needed, and social and economic benefits are increased.

Mesenchymal stem cells (Mesenchymal Stem Cells, MSCs) have the potential of multi-directional differentiation, can differentiate into various cells such as osteoblasts, adipocytes, fibroblasts, chondrocytes and the like, and are ideal seed cells for the current tissue engineering. Like other tissue-derived MSCs, bone marrow-derived mesenchymal stem cells (Bone marrow Mesenchymal Stem Cells, BMSCs) also have multiple differentiation capabilities. If autologous bone marrow mesenchymal stem cells are used for introducing into joint cavities for relevant treatment clinically, more possibilities are provided for the treatment of OA clinically.

More and more studies have shown that the inflammatory mechanism plays an important role in the development and progression of osteoarthritis, by modulating inflammatory cytokines and related signaling pathways, leading to degradation of extracellular matrix and dysfunction of chondrocytes, ultimately leading to the development of osteoarthritis. Our studies also demonstrate that significant increases in expression of IL-6 in joint fluids in OA animal models, combined with the recently emerging stem cell gene editing technology, would hold promise for stem cell directed differentiation therapies against osteoarthritis.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a gene editing mesenchymal stem cell and a preparation method and application thereof. The invention prepares a gene editing mesenchymal stem cell based on the significant increase of the expression of IL-6 in joint fluid in an OA animal model and the connection of STAT3 to UBE3A upstream by means of IL6/JAK/STAT3 signal pathway.

In order to achieve the above purpose, the technical scheme adopted by the invention is as follows: a method for preparing a gene editing mesenchymal stem cell, comprising the following steps: connecting a STAT3 promoter with the UBE3A fragment to construct a lentiviral expression vector; and transfecting the slow virus expression vector into the mesenchymal stem cells, and screening to obtain the gene editing mesenchymal stem cells. Janus Kinase/signal transduction and transcription activator (Janus Kinase/signal transducer and activator of transcription, JAK/STAT) is a signal pathway which is currently clinically studied more, and has a plurality of protein regulatory enzymes which mainly mediate signal transduction and gene transcription and activation, and finally mediate the effect of target gene activation. The present inventors have found that expression of OA rat joint fluid IL-6 is significantly elevated, resulting in STAT3 phosphorylation and promotion of nuclear entry. On the basis, the invention connects the STAT3 promoter to the upstream of UBE3A, constructs a p-STAT3-UBE3A vector, and transfects mesenchymal stem cells by adopting the vector to obtain the gene editing mesenchymal stem cell p-STAT3-UBE3A-MSC, and the cell has good repairing effect on osteoarthritis.

As a preferred embodiment of the method for preparing a gene-edited mesenchymal stem cell of the present invention, the mesenchymal stem cell is a bone marrow mesenchymal stem cell.

The invention also provides a gene editing mesenchymal stem cell, which is prepared by adopting the preparation method of the gene editing mesenchymal stem cell.

As a preferred embodiment of the gene-edited mesenchymal stem cell of the present invention, the gene-edited mesenchymal stem cell is differentiated into chondrogenesis under IL-6 or OA joint fluid stimulation.

As a preferred embodiment of the gene-editing mesenchymal stem cell of the present invention, UBE3A expression is up-regulated in the gene-editing mesenchymal stem cell.

As a preferred embodiment of the gene-edited mesenchymal stem cell of the present invention, METTL1 expression in the gene-edited mesenchymal stem cell is down-regulated.

The invention also provides a pharmaceutical composition for treating osteoarthritis, which comprises the gene editing mesenchymal stem cells and a pharmaceutically acceptable carrier.

The invention also provides application of the gene editing mesenchymal stem cells in preparing medicines for treating osteoarthritis.

The invention also provides application of the bone marrow mesenchymal stem cells knocked down with METTL1 in preparing a medicament for treating osteoarthritis.

The invention also provides application of the bone marrow mesenchymal stem cells over-expressing UBE3A in preparing a medicament for treating osteoarthritis. Ubiquitin-proteasome pathways are important mechanisms regulating a variety of cellular biological processes, mediating 80% -85% of protein degradation in eukaryotic organisms. While the key to mediating this process is ubiquitin-protein ligase (E3), UBE3A (E6-AP) is one of the important members of the ubiquitin-protein ligase (E3) family, which was initially found to have ubiquitin ligase activity that forms an E6/E6-AP protein complex with HPV 16-encoded E6 protooncoprotein, degrading the P53 protein via the ubiquitin-proteasome pathway, and subsequent studies have found that a large number of cellular proteins degrade via the E6-AP mediated ubiquitin-proteasome pathway, such as BAK, c-MYC, mcm-7, hScrib, HHR23A, SRC-3, and the like. The inventors of the present application have found that UBE3A promotes chondrogenic differentiation of BMSCs cells by modulating METTL1 degradation.

The invention has the beneficial effects that: the invention provides a gene editing mesenchymal stem cell, which is used for expressing UBE3A in high, promoting the ubiquitination of METTL1 and the degradation of METTL1 protein, promoting the differentiation efficiency of the mesenchymal stem cell to the cartilage forming direction and providing a new treatment way for repairing osteoarthritis cartilage.

Drawings

FIG. 1 shows the expression of METTL1 in control groups and BMSCs knocked down METTL1, wherein A is Western Blot to detect expression of METTL1 protein levels; b is qRT-PCR to detect mRNA level expression of METTL1, ACTB as an internal reference, n=3, P <0.001.

FIG. 2 shows the change in expression of chondrocyte markers during the chondrogenic differentiation of BMSCs of control group with WB assay knockdown METLL 1.

Fig. 3: a is co-immunoprecipitation confirming that METTL1 can interact with UBE 3A; b is western method to test UBE3A knock-down efficiency; c is western method to test UBE3A high expression effect; d shows up-regulation of METTL1 expression after UBE3A knockdown and down-expression of METTL1 after UBE3A high expression.

FIG. 4 is a graph showing changes in METTL1 protein levels following treatment with CHX protein synthesis inhibitor.

FIG. 5 is a graph showing the results of in vitro ubiquitination experiments.

FIG. 6 is a graph showing changes in the efficiency of induction of cartilage differentiation after UBE3A was expressed at a high level.

FIG. 7 is a schematic diagram of construction of UBE3A expression plasmid driven by STAT3 promoter, wherein A is UBE3A expression plasmid driven by STAT3 promoter (P-STAT 3-UBE 3A); b is WB evaluation of protein expression levels such as phospho-STAT 3, UBE3A and METTL1 under IL-6 stimulation or OA synovial fluid stimulation after transfection of P-STAT3-UBE 3A.

FIG. 8 is a graph showing the results of WB and Alxin blue staining.

Fig. 9 is a flow cytometry detection of Treg cell content in each group of peripheral blood; n=3, P <0.01.

FIG. 10 is a graph showing the proportion of Th17 cells in peripheral blood of osteoarthritis mice after treatment with MSC or p-STAT3-UBE3A-MSC.

FIG. 11 is a graph showing the proportion of M1 macrophages in joint fluid after flow cytometry detection in osteoarthritis mice receiving MSC treatment or p-STAT3-UBE3A-MSC treatment; n=3, P <0.01.

FIG. 12 is the effect of MSC and p-STAT3-UBE3A-MSC on the knee joint cartilage pathology of OA rats (HE staining, ×400); n=3, P <0.01, P <0.001.

Detailed Description

The above-described aspects of the present invention will be described in further detail below with reference to specific embodiments in the form of examples. It should not be understood that the scope of the above subject matter of the present invention is limited to the following examples only. All techniques implemented based on the above description of the invention are within the scope of the invention.

Example 1

In this example, experiments prove that knocking down METTL1 promotes differentiation of bone marrow mesenchymal stem cells to chondroblasts, and the specific experimental method is as follows:

(1) Isolation culture of primary mesenchymal stem cells: soaking 4-week-old male rat in 75% alcohol for 5min after cervical dislocation, taking out two-sided leg bones, removing muscle tissue in culture dish containing PBS solution, transferring into low-sugar DMEM containing 10% FBS, subtracting two ends of leg bones, repeatedly flushing bone marrow cavity with 1ml syringe, collecting culture solution, repeatedly blowing, standing for 10min, and transferring supernatant to 10ml aseptic centrifugationIn the tube, the supernatant was discarded after centrifugation at 3000rpm, and the cells were resuspended in low-sugar DMEM medium containing 10% FBS to adjust the cell density to 50X 10 ⁵ Inoculating in culture flask with 5% CO ₂ Culturing at 37deg.C, changing liquid every day and observing cell morphology; phenotype identification of mesenchymal stem cells: the third generation MSC was collected and cultured, washed once with 0.01mol/L PBS, and added with CD45, CD90, CD31, CD25, CD105, working concentration 1: after incubation for 30min at 100,4 ℃, PBS wash, fluorescein isothiocyanate labeled secondary antibody was added at a working concentration of 1: incubation is carried out for 30 minutes at 100 and 4 ℃, PBS is used for washing twice, and detection is carried out by a flow cytometer;

(2) Construction of BMSCs stable cell lines knocked down with METTL 1: the method comprises the steps of transfecting HEK-293T cells with packaging plasmids respectively by using pLKO.1-shMETTL1-1-Puro, pLKO.1-shMETTL1-5-Puro plasmid vectors and control pLKO.1-shGFP-Puro plasmid vectors which are constructed by teaching from a first hospital precision medical center Lin Shuibin affiliated to the university of Zhongshan, collecting virus liquid, infecting BMSCs, screening for about one week by puromycin, and performing expansion culture to obtain stable cell strains. mRNA and protein levels of METTL1 were detected by qRT-PCR and WB, respectively, and the expression level of METTL1 was found to be significantly reduced (as shown in FIG. 1) compared with that of control BMSCs, which showed that stable BMSCs cell lines knocked down with METTL1 were successfully constructed;

(3) The 3 rd generation of logarithmic growth phase bone marrow mesenchymal stem cells are taken, the experimental group uses a low-sugar DMEM culture medium containing 2mg/L insulin, 3mg/L transferrin, 1mmol/L pyruvic acid, 100nmol/L dexamethasone and 10 mu g/L transforming growth factor beta 1 to induce the differentiation of the human bone marrow mesenchymal stem cells into chondrocytes, and the cells of the control group are cultured by a low-sugar DMEM basic culture solution. Observing the morphology by an inverted microscope, inducing for 7-21 days, detecting the expression of SOX9 and COL2A1 by a western method, and analyzing the content of proteoglycan mucopolysaccharide by O-brilliant green staining of the saffron. The results are shown in FIG. 2, and the chondrocyte markers associated with the expression of chondrocytes induced to differentiate by BMSCs after knocking down METTL1 are significantly increased.

Example 2

Experiments prove that UBE3A promotes the chondrogenic differentiation of BMSCs cells by regulating and controlling the degradation of METTL1, and the specific experimental method is as follows:

(1) Co-immunoprecipitation verifies the interaction relationship of METTL1 with UBE 3A: mesenchymal stem cells were collected, washed twice with pre-chilled PBS, lysed for 30min at NP-40 degrees, incubated overnight with METTL1 antibody, and 100 l protein a (protein G or Sepharose) was added and gently shaken for 1h or overnight at 4 ℃ to capture immunoprecipitated complexes; agarose/Sepharose beads were collected by pulsed centrifugation (14000 rpm,5 s), the supernatant was discarded and the beads were washed 3 times with 800l ice-cold modified RIPA buffer; the agarose/Sepharose beads were resuspended in 60l of 2 XSDS protein loading buffer, and after mixing, boiled for 5min, centrifuged at 200g for 1min to discard the beads; the supernatant was transferred to a new centrifuge tube for cryopreservation at-20℃or SDS-PAGE.

(2) The pPB-CAG-UBE3A over-expressing UBE3A and the plasmid specifically knocked down UBE3A were transferred into BMSCs, respectively, and the transfection efficiency was examined with WB.

(3) Protein levels in control BMSCs, knock-down BMSCs of UBE3A, and BMSCs over-expressing UBE3A plasmid described above were detected with WB for METTL 1.

(4) Protein synthesis inhibitors, cycloheximide (CHX), were added to control BMSCs and to UBE3A knockdown BMSCs, and after 0h, 2h, 4h and 6h treatment, respectively, the change in METTL1 protein expression levels was verified with WB.

(5) In vitro ubiquitination experiment: mesenchymal stem cells highly express HA-METTL1 and Flag-UBE3A. After 72 hours, mesenchymal stem cells are centrifugally precipitated, and RIPA cell lysate is cracked; adding 10mM protease inhibitor and 10mM NEM; after super lysis and centrifugation, the supernatant was added with anti-HA antibody and agarose/Sepharose beads overnight at 4℃and washed, and then subjected to SDS electrophoresis and western detection.

(6) The BMSCs and the normal BMSCs which are used for highly expressing UBE3A are respectively used for inducing the chondrogenic differentiation, and then WB is used for detecting the protein level expression condition of the induced chondrocyte markers in each group.

The experimental results are shown in FIGS. 3 to 6. FIG. 3A shows that anti-METTL 1 antibodies can recruit to UBE3A protein, indicating the interaction between UBE3A and METTL 1; FIGS. 3B-C show that the UBE3A protein level of the BMSCs knocked down UBE3A is significantly reduced, and conversely, the over-expression plasmid successfully over-expresses UBE3A in the target cells, which indicates that the UBE3A over-expression and knockdown plasmid is successfully constructed; FIG. 3D shows that protein level expression of METTL1 can be enhanced after UBE3A silencing, and the above experimental results demonstrate that there is a correlation between UBE3A and METTL1 expression. As can be seen from fig. 4, the expression level of METTL1 in the control group gradually decreased with the prolonged action time of CHX, suggesting that CHX prevents the synthesis of METTL1 protein; however, in the UBE3A knockdown group, protein stability of METTL1 did not change significantly. As can be seen from fig. 5, in BMSCs that co-express METTL1 with UBE3A, UBE3A protein expression promotes ubiquitination degradation of METTL1 protein; METTL1 ubiquitination was significantly reduced in BMSCs silencing UBE3A expression. These results demonstrate that METTL1 is a substrate for UBE3A, and UBE3A can promote ubiquitination degradation of METTL 1. As can be seen from FIG. 6, the chondrocyte expression-related chondrocyte markers of BMSCs induced differentiation after high UBE3A expression are increased.

Example 3

The embodiment provides a preparation method of a gene editing mesenchymal stem cell, which comprises the following steps:

(1) The STAT3 promoter sequence was ligated with the indicator gene UBE3A by OE-PCR using a lentiviral backbone vector and inserted into the plasmid polyclonal site. Transformation competence, colony selection, plasmid amplification by shaking, plasmid extraction using plasmid mass extraction kit. And co-transfecting 293T cells with core plasmids and lentivirus helper plasmids phelper1 and phelper2, collecting virus supernatant after 3 days to infect mesenchymal stem cells, and screening puromycin after the infection of the mesenchymal stem cells to obtain the gene editing mesenchymal stem cells p-STAT3-UBE3A-MSC.

(2) The experimental group was treated with 500pg of IL6 for 24 hours, the control group was not treated, and the expression levels of UBE3A and METTL1 were evaluated using WB.

As shown in FIG. 7, only under the stimulation of IL-6 or OA synovial fluid after transfection of P-STAT3-UBE3A, STAT3 can be phosphorylated to promote high expression of UBE3A, and METTL1 expression is reduced.

(3) Under the stimulation of IL-6, wild type mesenchymal stem cells and p-STAT3-UBE3A-MSC are induced to form cartilage, and the level of differentiation of the mesenchymal stem cells into the cartilage is evaluated by an aliskiren blue staining method and WB.

As shown in the experimental result in FIG. 8, compared with the wild type mesenchymal stem cells, the chondrocyte expression related chondrocyte markers induced and differentiated by the P-STAT3-UBE3A-MSC are obviously increased, and similarly, the Alxin blue staining shows that the chondrocyte differentiation induction efficiency of the transfected P-STAT3-UBE3A is obviously improved under the action of IL-6.

Example 4

The protection mechanism of p-STAT3-UBE3A-MSC on osteoarthritis and the influence on CD4+ T cell subsets are experimentally explored in the embodiment, and the specific experimental method is as follows:

(1) Establishment of knee osteoarthritis model: 40 adult SD male rats, weighing (280+ -20) g, were anesthetized with chloral hydrate (40 mg/kg, intraperitoneal injection) after pain threshold detection prior to molding, and 0.3mg of monomer iodoacetic acid (monomer sodium iodic acid, MIA) solution was injected into the left knee joint cavity of the rats, in combination with continued drawer-like joint movement. The specific operation steps are that 3mg MIA is dissolved in 50mL of 0.9% physiological saline, the mouse hair near the joint is cleaned, the joint is flexed to the maximum extent, the prepared solution is injected into the left knee joint cavity, and the same dose of physiological saline is injected into the right knee. The normal group was injected with 50mL of physiological saline into both knees in the same manner. All mice were simultaneously subjected to the same driving exercise for about 30min each day. 15d after successful molding, p-STAT3-UBE3A-MSC is injected into the joint cavity of the experimental group, wild mesenchymal stem cells (MSC group) are injected into the joint cavity of the control group, and no intervention is performed in the normal group (OA group). And weighing 43d after molding, and testing the mechanical foot shrinkage threshold of pain behavioural before and after molding. The percentage of Treg cells in the peripheral blood of each group of mice to CD4+ cells and the proportion of Th17 cells were examined.

The results are shown in FIG. 9, the percentage of Treg cells in the peripheral blood of the MSC group and the p-STAT3-UBE3A-MSC group to CD4+ cells is obviously higher than that of the OA group, and the difference is statistically significant; the differences were not statistically significant in the MSC group compared to the p-STAT3-UBE3A-MSC group, indicating that the clear proportion of regulatory T cells in peripheral blood was significantly increased after either MSC treatment or p-STAT3-UBE3A-MSC treatment in osteoarthritis mice.

As shown in FIG. 10, there was no significant change in the proportion of Th17 cells in peripheral blood after MSC treatment or p-STAT3-UBE3A-MSC treatment in osteoarthritis mice, nor was there any statistical significance in the difference between the MSC group and the p-STAT3-UBE3A-MSC group, indicating that there was no significant change in Th17 cells in peripheral blood after MSC treatment or p-STAT3-UBE3A-MSC treatment in osteoarthritis rats.

(2) After the synovial fluid of the joint is obtained, the complete culture medium is diluted, the synovial fluid is centrifuged for 20 minutes at room temperature by 500g, nucleated cells of the synovial fluid are obtained by Ficoll-Hypaque fluid layering and centrifugation, the complete culture medium is washed, and the antibody is incubated for detection by a flow cytometer. Preparation of articular subchondral bone single cell suspension, tibial plateau PBS washing, separation of cartilage and subchondral bone under dissecting microscope, sagittal chopping of bone to one millimeter, digestion in 8 ml aMEM with 0.6mg/ml collagenase IA, rotary stirring digestion at 37℃for 4 hours. After digestion, the suspension was screened through a 70 m screen, and on-machine assessed for diagonal effects, if any, FVD or 7-AAD staining was used to identify dead-alive to eliminate diagonal effects, if not, paraformaldehyde fixation was used directly, and the specimens were spun down and washed 3 more times with PBS before staining. The changes in the content of M1 type macrophages in the joint fluid of each group of mice were examined.

As shown in fig. 11, the proportion of M1 macrophages in the joint fluid of each OA-treated group was significantly reduced compared to the peripheral blood itself. The percentage of M1 macrophages in joint fluid of both MSCs and p-STAT3-UBE3A-MSC treated groups was significantly lower than that of untreated OA control groups, and the differences were not statistically significant compared to the MSC and p-STAT3-UBE3A-MSC groups, indicating a significant decrease in the proportion of M1 macrophages in joint fluid after MSC treatment or p-STAT3-UBE3A-MSC treatment in osteoarthritis rats.

(3) After the rat is sacrificed, obtaining the tibial subchondral bone, cutting the frozen section into 10 mu m, placing the frozen section in a 37 ℃ oven for 60min, and drying the frozen section by moisture; rinsing with PBS for 3 times, each time for 10min, throwing away and wiping off liquid around the tissue, and placing the slice in a wet box; surrounding the tissue with an immunohistochemical pen, blocking in a wet box at 37℃for 30min with 5% BSA/PBS; excess liquid was removed and 100ul of anti-RANKL antibody (diluted with 5% BSA 1:100) was added dropwise to the tissue overnight at 4 ℃. The next day, the sections were washed 3 times with PBS for 10min each time; spin-drying the residual liquid, adding a fluorescent secondary antibody (diluted by 5% BSA 1:100), and incubating for 1h at 37 ℃ in a dark place; PBS is washed for 3 times, each time for 10min; and observing under a fluorescence microscope.

As shown in FIG. 12, the OA group rats had severe cartilage surface damage, defect or disappearance of the tidal line, morphological destruction of chondrocytes, a significant decrease in the number and an increase in cell diffusion. MSCs and p-STAT3-UBE3A-MSC treated rats are significantly improved in knee joint cartilage destruction. The OARSI scoring result shows that compared with the untreated OA control group, the MSCs and the p-STAT3-UBE3A-MSC have obviously reduced cartilage cell destruction degree after the OA rat is treated by the OA control group, and particularly the p-STAT3-UBE3A-MSC treatment group has better effect.

Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made to the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. A method for preparing a gene editing mesenchymal stem cell, which is characterized by comprising the following steps:

connecting a STAT3 promoter with the UBE3A fragment to construct a lentiviral expression vector; and transfecting the slow virus expression vector into the mesenchymal stem cells, and screening to obtain the gene editing mesenchymal stem cells.

2. The method for preparing a genetically edited mesenchymal stem cell of claim 1, wherein the mesenchymal stem cell is a bone marrow mesenchymal stem cell.

3. A genetically edited mesenchymal stem cell prepared by the method of any one of claims 1 to 2.

4. The genetically engineered mesenchymal stem cell of claim 3, wherein the genetically engineered mesenchymal stem cell is differentiated to chondrogenesis under IL-6 or OA joint fluid stimulation.

5. The genetically engineered mesenchymal stem cell of claim 3, wherein UBE3A expression is upregulated in the genetically engineered mesenchymal stem cell.

6. The genetically engineered mesenchymal stem cell of claim 3, wherein METTL1 expression is down-regulated in the genetically engineered mesenchymal stem cell.

7. A pharmaceutical composition for the treatment of osteoarthritis comprising the genetically engineered mesenchymal stem cell of any one of claims 3-6 and a pharmaceutically acceptable carrier.

8. Use of the genetically edited mesenchymal stem cell of any one of claims 3-6 in the preparation of a medicament for treating osteoarthritis.

9. Use of bone marrow mesenchymal stem cells knocked down with METTL1 in the preparation of a medicament for treating osteoarthritis.

10. Use of bone marrow mesenchymal stem cells overexpressing UBE3A in the preparation of a medicament for treating osteoarthritis.