Effects Of HOXC10 Gene On Proliferation,Apoptosis And Differentiation Of Sheep Bone Marrow Mesenchymal Stem Cells

Bone marrow mesenchymal stem cells(BMSCs)which are derived from mesoderm are important adult stem cells.BMSCs are able to differentiate into a variety of cell types,such as osteoblasts,chondrocytes,adipocytes and so on.It is well known that BMSCs play vital role in tissue regeneration.However,the molecular mechanism of directing differentiation of BMSCs remains elusive.It is unclear which kinds of factors affect its multipotency.Recent studies have found that the Homeobox(HOX)genes are involved in the regulation of proliferation,apoptosis and differentiation of BMSCs.In this study,we isolated sheep BMSCs(sBMSCs),and constructed HOXC10 overexpression vector and HOXC10 interference vector,respectively.To investigate the impact of HOXC10 on proliferation and apoptosis,sBMSCs were transfected with HOXC10 overexpression vector and HOXC10 interference vector,respectively.Then,adipogenic induction and osteogenic induction were performed to investigate the function of HOXC10 in the process of adipogenic and osteogenic differentiation of sBMSCs,and explore its effect on cells differentiation.BMSCs were successfully isolated by cell adherence method,and their multipotency were characterized by passage,immunofluorescence and induction.At the same time,the overexpression vector and RNAi vector of HOXC10 were successfully constructed by PCR,restriction enzyme digestion,product recycle and ligation,and so on.The sBMSCs were electroporated at 220 V and 2.5ms,and the efficiency was detected 48 h after electroporation.The result of qPCR showed that the expression level of HOXC10 was increased by 371.68 times than that of the control group after transfecting HOXC10 overexpression vector into sBMSCs.In contrast,the expression level of HOXC10 in sBMSCs was reduced by 80.38% compared with the control group after transfecting HOXC10 RNAi vector into sBMSCs.After electroporation,sBMSCs were cultured for 24 h,48h and 72 h,CCK8 was respectively added,and the absorbance value was detected at 450 nm.It was demonstrated overexpression of HOXC10 promoted cell growth,while knockdown of HOXC10 inhibited cell growth.Flow cytometry was used to detect the effect of HOXC10 on apoptosis of sBMSCs.It was found that HOXC10 overexpression could inhibit the apoptosis of sBMSCs,while knockdown of HOXC10 could promote the apoptosis of sBMSCs.The adipogenesis of sBMSCs which were respectively transfected with overexpression vector and RNAi vector of HOXC10 were detected by oil red O staining and lipid titration detection after 3,6,12 and 18 day of adipogenic induction.The mRNA transcription level of LPL,PPARG,Fabp4 and ACC were detected by qPCR.The protein expression level of LPL and PPARG were detected by Western Blot and gray scale analysis.It was demonstrated that HOXC10 overexpression promoted adipogenic induction and interfered with HOXC10 inhibit adipogenesis.The osteogenesis of sBMSCs which were transfected with overexpression vector and RNAi vector of HOXC10 were detected by alizarin red staining and titration detection of steogenesis induced at 3,7,11 and 14 day,respectively.The mRNA transcription level of COLIA1,BMP5,Runx2,OPN were analyzed by qPCR.The protein expression level of Runx2,OPN were analyzed by Western Blot and gray scale,respectively.It was showed that HOXC10 gene overexpression and knockdown had no obvious effect on osteogenesis of sBMSCs.The present study confirmed that HOXC10 overexpression could promote the proliferation of sBMSCs,inhibited its apoptosis,and promoted adipogenesis.Conversely,HOXC10 knockdown could inhibit the proliferation of sBMSCs,promoted its apoptosis,and inhibited adipogenesis.Our study found that the expression of HOXC10 in sBMSCs had no significant effect on osteogenesis.In sum,our study provided an experimental basis for further study on the function of HOXC10 in the differentiation process of BMSCs and its mechanism of differentiation potential.