The Regulatory Mechanism Of LncRNA CIR In The Chondrogenic Differentiation From Umbilical Cord Mesenchymal Stem Cells

Objective: Osteoarthritis(OA),characterized by irreversible cartilage damage,remains one of the most prevalent orthopedic diseases that severely affects motor function and life quality of patients.Tissue engineering methods have been considered the promising technology to repair the damaged cartilage which is extensively investigated in the OA area..However,the insufficient source of seed cells limits its clinical application to some extent.Due to its easily accessible,The reliable chondrogenic capacity of human umbilical cord mesenchymal stem cells(hUC-MSCs)is essential for the cartilage regeneration.In this study,we investigated the regulatory mechanism of lncRNA CIR underlying chondrogenic differentiation of hUC-MSCs,providing new evidence for theapplication of hUC-MSCs in the therapy of OA.Methods:(1)hUC-MSCs were extracted from the umbilical cord of healthy maternity and their cellular morphology was observed under a microscope.The surface markers of hUC-MSCs were analyzed by flow cytometry.Alizarin red S,Alcian blue,and Oil Red O staining were used to examine the osteogenic,chondrogenic,and adipogenic differentiation abilities of primary isolated hUC-MSCs after incubation with the specific induction medium.(2)The expression level of lncRNA CIR at various time points during chondrogenic differentiation of hUC-MSCs was evaluated by q PCR.si RNAs targeting lncRNA CIR(si-lncRNA CIR)were designed and transfected into hUC-MSCs,and the interference efficiency was validated by q PCR.Alcian blue staining was adopted to detect the chondrogenic differentiation ability of hUC-MSCs before and after knockdown of lncRNA CIR.To evaluate the effect of lncRNA CIR silencing on hUC-MSC chondrogenic differentiation,Western blotting and immunofluorescence staining were performed to measure the protein levels of chondrogenic differentiation markers(SOX9,Aggrecan,and Col2A1).(3)The expression level of ATOH8 at different time points during hUC-MSC chondrogenic differentiation was assessed by q PCR and Western blotting.The overexpression plasmid for ATOH8 was constructed and transfected into hUC-MSCs to overexpress ATOH8 in hUC-MSCs.The chondrogenic differentiation ability of hUC-MSCs was measured by Alcian blue staining,and the protein levels of chondrogenic differentiation markers(SOX9,Aggrecan,and Col2A1)were determined by Western blotting and immunofluorescence staining,so as to examine the effect of ATOH8 overexpression on hUC-MSC chondrogenic differentiation.(4)The mRNA and protein levels of ATOH8 in lncRNA CIR overexpressed-or depleted-hUC-MSCs were detected by q PCR and Western blotting to examine the regulation of lncRNA CIR in ATOH8 expression.The expression level of lncRNA CIR in the nucleus and cytoplasm was detected by q PCR after nuclear and cytoplasmic separation.RIP and RNA pull-down assays were carried out to verify the interaction between EZH2/H3K27me3 and lncRNA CIR.After silencing or overexpressing EZH2,the protein levels of EZH2,ATOH8 and H3K27me3 were detected by Western blotting.The enrichment of EZH2 or H3K27me3 in the promoter region of ATOH8 was determined by Ch IP/q PCR.To evaluate whether lncRNA CIR promoted the methylation inhibition of ATOH8 through EZH2,the EZH2-deficient hUC-MSCs were transfected with overexpression plasmid of lncRNA CIR,and then the protein levels of ATOH8 and H3K27me3 were assessed by Western blotting.(5)hUC-MSCs were co-transfected with si-ATOH8 and si-lncRNA CIR to examine the modulation of lncRNA CIR/ATOH8 axis in hUC-MSC chondrogenic differentiation.The changes in chondrogenic differentiation ability were detected by Alcian blue staining.Protein expression levels of chondrogenic differentiation markers(SOX9,Aggrecan,and Col2A1)were measured by Western blotting and immunofluorescence staining.Results:(1)The spindle-shaped hUC-MSCs were observed under a microscope.High expression of CD90 and CD44,while lack of expression of CD45 and CD34 in hUC-MSCs were validated by flow cytometry.The osteogenic,chondrogenic and adipogenic differentiation capacities of hUC-MSCs were confirmed,indicating the successful isolation of hUC-MSCs.(2)The expression of lncRNA CIR was gradually decreased with the prolonging of time during hUC-MSC chondrogenic differentiation.Knockdown of lncRNA CIR increased chondrogenic differentiation ability as evidenced by up-regulating SOX9,Aggrecan,and Col2A1.(3)ATOH8 level was gradually elevated during chondrogenic differentiation of hUC-MSCs.Overexpression of ATOH8 enhanced chondrogenic differentiation ability and the protein levels of SOX9,Aggrecan,and Col2A1.(4)Depletion of lncRNA CIR significantly raised the mRNA and protein levels of ATOH8.LncRNA-CIR was mainly located in the nucleus.Knockdown of EZH2 led to down-regulation of H3K27me3,but up-regulation of ATOH8.As expected,opposite results were obtained after overexpression of EZH2.EZH2 and H3K27me3 directly bound to the promoter region of ATOH8,which was weakened by lncRNA CIR silencing.Overexpression of lncRNA CIR did not affect ATOH8 expression in EZH2-depleted cells.(5)The promotive effects of lncRNA CIR deficiency on hUC-MSC chondrogenic differentiation and expression of SOX9,Aggrecan,and Col2A1 were counteracted by ATOH8 inhibition.Conclusion: This study found that lncRNA CIR was down-regulated,but ATOH8 was up-regulated during the chondrogenic differentiation of hUC-MSCs.Functional experiments indicated that lncRNA CIR inhibited hUC-MSC chondrogenic differentiation,while ATOH8 exerted the opposite roles.Mechanistically,lncRNA CIR recruited EZH2 to ATOH8 promoter to repress the transcription of ATOH8.After knockout of EZH2,lncRNA CIR-mediated regulation of ATOH8 expression and hUC-MSC chondrogenic differentiation was abolished.