| Background:Osteogenic differentiation of human bone marrow mesenchymal stem cells(hBMSCs)portrays a crucial role in the process of bone formation and development.In recent years,accumulating studies has confirmed that long non-coding RNA(lncRNA)are involved in osteogenic differentiation of hBMSCs.However,their exact functions and molecular mechanisms in osteogenic differentiation remain largely elusive.As a consequence,the purpose of this study is to elucidate the function and mechanism of KCNMA1-AS1 in osteogenic differentiation of hBMSCs.Methods:hBMSCs were cultured in growth medium for proliferation and osteogenic medium for osteogenic induction,the expression levels of KCNMA1-AS1 and osteogenesis-related genes during osteogenic differentiation of hBMSCs were measured by quantitative real-time polymerase chain reaction(qPCR),the protein levels of osteogenic specific markers during osteogenesis of hBMSCs were detected by Western blot,Alkaline phosphatase(ALP)staining and ALP activity assay as well as Alizarin Red S(ARS)staining and ARS quantification were performed to determine the osteogenic potential of hBMSCs.Besides,correlation analysis was employed to investigate the association between the expression level of KCNMA1-AS1 and osteogenesis-related genes.KCNMA1-AS1 overexpression and KCNMA1-AS1 knockdown lentivirus were constructed and transfected into hBMSCs.In vitro experiments including qPCR,Western blot,ALP staining and ARS staining together with in vivo experiments were deployed to explore the effect of KCNMA1-AS1 on osteogenic differentiation of hBMSCs.Then,fluorescence in situ hybridization(FISH)assay was conducted to examine the subcellular localization of KCNMA1-AS1 in hBMSCs.SMAD9 was screened out to be the binding protein with KCNMA1-AS1 by applying RNA pull-down assay and mass spectrometry,RNA binding protein immunoprecipitation(RIP)assay was used to confirm the interaction between KCNMA1-AS1 and SMAD9.In addition,the expression levels of total SMAD9 and phosphorylated SMAD9 were detected by Western blot in KCNMA1-AS1 overexpression or KCNMA1-AS1 knockdown transfected hBMSCs.Moreover,rescue assays were performed using LDN193189 as the inhibitor of SMAD9 signaling pathway,the protein levels of osteogenic specific markers in NC or KCNMA1-AS1 overexpression transfected hBMSCs treated with LDN193189 were detected by Western blot,ALP staining and ARS staining were used to evaluate the osteogenic differentiation of hBMSCs.Results:The expression level of KCNMA1-AS1 was significantly increased during osteogenic differentiation of hBMSCs,the expression levels of osteogenesis-related genes were also upregulated.Meanwhile,ALP activity and calcium nodules deposition were distinctly improved after osteogenic induction,which indicated that hBMSCs have potential of osteogenic differentiation towards osteoblasts.Positive correlation was discovered between the expression levels of KCNMA1-AS1 and osteogenesisrelated genes COL1A1,RUNX2 and OSX.Gain and loss function assays performed with lentivirus transfection revealed that KCNMA1-AS1 overexpression promoted osteogenic differentiation of hBMSCs in vitro and enhanced bone formation in vivo,whereas KCNMA1-AS1 knockdown presented the contrary.FISH assay demonstrated that KCNMA1-AS1 was mainly distributed in the nucleus of hBMSCs.The effective combination between KCNMA1-AS1 and SMAD9 was confirmed by RNA pull-down,mass spectrometry and RIP assays.Also,KCNMA1-AS1 overexpression triggered the phosphorylation of SMAD9,therefore activated SMAD9 signaling pathway.Furthermore,rescue assays unveiled that LDN193189 as the inhibitor of SMAD9 signaling pathway counteracted the stimulative effects of KCNMA1-AS1 overexpression on osteogenic differentiation of hBMSCs.Conclusions:Taken together,the findings above evidenced that KCNMA1-AS1 directly binds to SMAD9 and promotes osteogenic differentiation of hBMSCs by activating the SMAD9 signaling pathway,which has potential to serve as a biomarker and therapeutic target for restoration of bone defect. |
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