The Role Of MicroRNA-143 In Chondrogenic Differentiation Of Synovial Mesenchymal Stem Cells Induced By Cyclic Tensile Stress

Objectives:This study aims to investigate the role of micro RNA in the chondrogenic differentiation of synovial mesenchymal stem cells induced by cyclic tensile stress and its mechanism.Methods:1.Rat mandibular extension model was established,and then condylar cartilage specimens were collected at 4 time points after the successful establishment of the model: day 3,week 1,week 2,and week 3.The changes of synovium of TMJ in rats compared with NC group were observed by hematoxylin/eosin(HE)staining.The expression levels of chondrogenic genes such as SOX9 and COL2,and Rankl was detected by Immunofluorescence technique.2.The purchased human synovial mesenchymal stem cells were identified by flow cytometry after resuscitation and passage,and the main identification indexes were CD8,CD34,CD44 and CD90.The multidirectional induction and differentiation of synovial mesenchymal stem cells into osteogenesis,chondrogenesis and lipids was conducted to study the multidirectional induction and differentiation ability of synovial mesenchymal stem cells.3.In vitro mechanical stimulation model was constructed.Under the condition of0.5Hz force-loading frequency,the model synovial mesenchymal stem cells were divided into control group,5% force-loading group and 10% forceloading group.4.Screening and research on differential expression of micro RNA in vitro.Total RNA was extracted for high-throughput sequencing of the total RNA extracted from the SCSCS for 24 h,and micro RNAs with differential expression were screened out: miRNA-143,miR-499a-5p,miRNA129-5p,miRNA-335-3p,and miRNA-206,and the accuracy of high-throughput sequencing results was verified.5.Verify the effect of miR-143 on chondrogenic differentiation of human SMSCs.The overexpression and inhibition of miR-143 virus transfection system were constructed,and the effect of CTS on chondroblast differentiation was investigated.Grouping: The six groups of NC mimic,miR-143 mimic,miR-143 mimic+5%CTS,NC inhibitor,miR-143 inhibitor,miR-143inhibitor+5%CTS were added with chondroblast differentiation inducer.After72 hours of treatment,the relative protein expression levels of SOX9,ACAN and COL2 were detected by Western Blot.After 72 h,each group was stained with alcian blue and toluidine blue.6.The target gene of miR-143 was predicted through bioinformatics websites to study the influence of miR-143 target gene expression level.Results:1.Inflammatory changes occurred in condyle synovium of mandibular extension model in rats.Inflammatory changes began to occur in the 3d group,and decreased in the 1st and 2nd week groups,while increased in the 3rd week group.Inflammatory changes appear in the mandibular front,then repair themselves,decrease inflammatory changes,and increase inflammatory changes again beyond a certain limit.2.Compared with the control group,synovial chondrogenic activity was enhanced and osteoclast activity was weakened in the mandibular extension model rats at week 2.It is proved that mandibular anterior leads can stimulate synovial chondrogenic activity and inhibit osteoclast activity within a certain limit.3.By studying the induction of multidirectional differentiation of synovial mesenchymal stem cells and the effect of cyclic tensile stress on chondrogenic differentiation of synovial mesenchymal stem cells,the multidirectional differentiation ability of synovial mesenchymal stem cells was proved,and the in vitro augmentive model of synovial mesenchymal stem cells was successfully constructed.It is proved that 5% CTS can promote chondrogenic differentiation of synovial mesenchymal stem cells.4.High-throughput sequencing technology was applied to screen out and verify differentially expressed miRNAs of synovial-mesenchymal stem cells in chondrogenic differentiation induced by cyclic tensile stress,and miR-143 was selected as the research object to prove that miR-143 is stress-sensitive miRNA.It was proved that miR-143 expression was decreased in synovial mesenchymal stem cells induced by 5% CTS.5.miR-143 was selected as the research object to study the role of miR-143 in chondrogenic differentiation induction of synovial mesenchymal stem cells.The expression of miR-143 was reduced during chondrogenic differentiation of synovial mesenchymal stem cells,and miR-143 inhibited chondrogenic differentiation of synovial mesenchymal stem cells induced by cyclic tensile stress.According to the predicted target genes,it is speculated that miR-143 may inhibit chondrogenic differentiation of synovial-mesenchymal stem cells induced by cyclic tension stress through BMPR2.Conclusion:In this study,the mandibular extension model of rats was established.Pathological analysis and score of HE staining and immunohistochemical staining showed that in the mandibular extension model of rats,synovial tissue may be involved in chondrogenic differentiation during stress.Through culture identification and multidirectional differentiation induction of SMSCs,it was proved that SMSCs had chondroblast differentiation ability.Stress sensitive gene miR-143 was screened by in vitro mechanical stimulation model construction and high-throughput sequencing.The conclusion that miR-143 can inhibit the chondrogenic differentiation of SMSCs induced by CTS was obtained by PCR,Western Blot and other techniques,BMPR2 was also reserved as the target gene of miR-143 through bioinformatics,and it is speculated that miR-143 may regulate the chondrogenic differentiation of SMSCs induced by CTS by targeting BMPR2.