| Research Background:With the rise of national sports,tendon injury has become a very common sports injury.At present,the clinical treatment of tendon injury has some limitations such as long recovery cycle,high recurrence rate and unable to restore the original biomechanical properties.In recent years,more and more evidences show that tendon stem cells(TSCs)have the potential of self-renewal and multi-differentiation to repair damaged tendon tissue.At present,some studies have pointed out that the abnormal differentiation of TSCs is the basis of tendon injury.Therefore,it is crucial to clarify whether inhibiting the abnormal differentiation of TSCs can improve the degree of tendon injury.Long non coding RNA(lncrna)and microRNA(miRNA)can regulate each other with multiple sites and targets,and participate in cell differentiation,proliferation and the occurrence and development of many diseases.The role of miRNA in the multi-directional differentiation of mesenchymal stem cells has become a hot topic.Studies have shown that miR-138 can affect the adipogenic and osteogenic differentiation of mesenchymal stem cells by regulating the expression of peroxisome proliferator activated receptor gamma(PPARy)and Runx family transcription factor 2(Runx2);LncRNA KCNQ1 opposite strand/antisense transcript 1(kcnqlotl)may play a role in osteogenic differentiation and participate in the regulation of osteolysis,and It has been reported that LncRNA KCNQ1OT1 can play the role of endogenous miR-138.However,whether LncRNA KCNQ1OT1 can regulate the osteogenic and adipogenic differentiation of TSCs through miR-138 remains unclearTherefore,this study will explore the role of LncRNA KCNQ1OT1 and miR-138 in the adipogenic and osteogenic differentiation of TSCs,So as to provide new ideas and molecular theoretical basis for clinical treatment of tendon injury.Objective:1.To explore the effect of TSCs injection on the repair injured tendon tissue and to investigate the expression levels of LncRNA KCNQ1OT1 and miR-138 in vivo.2.To explore the role of LncRNA KCNQ1OT1 and miR-138 in adipogenic and osteogenic differentiation of TSCs3.To explore whether LncRNA KCNQ1OT1 could combine with miR-138 in vitro experiment.3.To demonstrate the effects of LncRNA KCNQ1OT1 in the adipogenic differentiation and osteogenic differentiation of TSCs and its inhibitory effect on the repair of injured tendon in vivo.Methods:1.A mouse tendon injury model was established.The maximum load and stiffness of the mouse Achilles tendon tissue were tested by biomechanics.The expression levels of LncRNA KCNQ1OT1 and miR-138 in tendon tissues were detected by real-time quantitative PCR(qRT-PCR)and the protein levels of RUNX2 and PPARy in tendon tissues were analyzed by Western blot.2.TSCs were cultured in vitro.The expression of the surface markers of TSCs were detected by flow cytometry.Then,TSCs were Then cultured in adipogenic and osteogenic media respectively,and Oil red O staining and alizarin red staining were used to observe the rnulti-directional differentiation ability of cells,and the levels of transcription factors related to adipogenic differentiation and osteogenic differentiation were measured by Western blot.qRT-PCR was used to detect the expression of LncRNA KCNQ1OT1 and miR-138 in TSCs induced by adipogenic differentiation and osteogenic differentiation.After interference with LncRNA KCNQ1OT1 in TSCs,qRT-PCR was performed to detect the relative adiponectin levels,Western blot was used to analyze the protein levels of PPAR?,RUNX2 and Osterix,and the activity of alkaline phosphatase(ALP)was measured.3.Bioinformatics prediction(Diana and RNAinter)found that there was a binding site between LncRNA KCNQ1OT1 and miR-138,and the interaction between LncRNA KCNQ1OT1 and miR-138 was further clarified by RNA immunoprecipitation(RIP)and RNA pull-down assays.4.After transfection of si-KCNQ1OT1 in vitro,the expression of miR-138 in TSCs was detected by qRT-PCR;Then,si-control,si-KCNQ1OT1,si-KCNQ1OT1+NC,and si-KCNQ1OT1+miR-138 inhibitor was transfected into adipogenic and osteogenic differentiation-induced TSCs in vitro respectively,qRT-PCR was used to detect the expression of miR-138 and the relative adiponectin levels,and Western blot was performed to analyze the protein levels of PPARy,RUNX2 and Osterix.4.A mouse model of tendon injury in vivo was established,then The TSCs transfected with pcDNA-KCNQ1OT1 and empty vector(pcDNA)were injected into the injured tendon,The maximum load and stiffness of the mouse tendon tissue were evaluated by biomechanics.qRT-PCR was used to detect the expression of miR-138 in mouse tendon tissues.Western blot was performed to analyze the protein levels of RUNX2 and PPAR? in mouse tendon tissues.Results:1.Biomechanical test results show that the maximum load and stiffness of mouse tendon tissues were increased in the Injured+TSCs group compared with the Injured group(P<0.05).qRT-PCR results showed that TSCs could down-regulated the expression of LncRNA KCNQIOT1 and up-regulated the expression of miR-138.The protein levels of RUNX2 and PPARy tested by Western blot in were up-regulated in the injured group compared with the control group.It is suggested that injection of TSCs can protect the injured tendon.2.Flow cytometry confirmed that the isolated cells were TSCs.Oil red O staining and alizarin red staining confirmed that TSCs had the potential of adipogenic and osteogenic differentiation.Western blot analysis showed that the expression of adipogenic and osteogenic proteins was up-regulated after TSCs were induced in adipogenic and osteogenic differentiation medium;qRT-PCR analysis showed that the expression of LncRNA KCNQ1OT1 was significantly increased,while the expression of miR-138 was significantly decreased(P<0.05).After interfering with the expression of LncRNA KCNQ1OT1 in TSCs,all the above expression levels were reversed.3.RIP assay showed that miR-138 and LncRNA KCNQIOT1 were enriched in the immunoprecipitation complex of ago2 antibody compared with input group;RNA pull-down assay showed that miR-138 was enriched in the pull-down complex of LncRNA KCNQ1OT1.Both of them revealed the interaction between lncrna kcnqlotl and miR-1384.After LncRNA KCNQIOT1 was interfered in TSCs,the expression of miR-138 tested by qRT-PCR was up-regulated,while the protein levels of PPARy,Runx2 and osterix tested by Western blot were down regulated(P<0.05),and these changes were reversed by miR-138 inhibitor.It indicated that the expression level of LncRNA KCNQ1OT1 was negatively correlated with miR-138.5.Biomechanical test results showed that the maximum load and stiffness of the tendon tissue of mice in the over-expressed LncRNA KCNQ1OT1 group(Injured+pcDNA-KCNQ1OT1-TSCs)were decreased(P<0.05).The expression of miR-138 tested by qRT-PCR was significantly down-regulated and the levels of Runx2 and PPARy tested by Western blot analysis were up-regulated in the iInjured+pcDNA-KCNQ1OT1-TSCs respectively(P<0.05).The results showed that LncRNA KCNQ1OT1 could affect the adipogenic and osteogenic differentiation of TSCs,which was an unfavorable factor for tendon healing.Conclusion:1.The expression of LncRNA KCNQ1OT1 was up-regulated in the injured tendon tissue,but the level of LncRNA KCNQ1OT1 was significantly decreased and the biomechanical properties of the injured tendon tissue were improved after injection of TSCs.2.LncRNA KCNQ1OT1 is involved in the regulation of adipogenic and osteogenic differentiation of TSCs.Interfering with the expression of LncRNA KCNQ1OT1 can weaken the osteogenic and adipogenic differentiation of TSCs.3.In vitro,it was confirmed that LncRNA KCNQ1OT1 could combine with miR-138,and it could negatively regulate miR-138.4.LncRNA KCNQ1OT1 can promote adipogenic and osteogenic differentiation of TSCs and inhibit tendon healing through miR-138. |
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