The Effects Of MiR-320 On The Proliferation And Differentiation In Human Alveolar Bone-derived Mesenchymal Stem Cells

Objectives This study aims to investigate the effect of miR-320 on the proliferation and osteogenic differentiation of human alveolar bone-derived mesenchymal stem cells(ABBMSCs),as well as on the expression of osteogenesis-related genes.This study will provide basis for clarify the molecular mechanism of alveolar bone reconstruction.Methods 1 Human AB-BMSCs were isolated and cultured by tissue block method.Then,the AB-BMSCs were identified by cellular Immunofluorescence.2 The AB-BMSCs were transfected with miR-320 mimics for 24 hours.Then,CCK-8,microplate assay and Alizarin Red were used to detect the effects of miR-320 on proliferative activity,intracellular ALP protein activity and osteogenic ability.RT-q PCR was used to analyze the m RNA levels of osteogenesis-related genes(HOXA10,Runx2,BGP,COL-I,ALP).3 The AB-BMSCs were transfected with miR-320 inhibitor for 24 hours.Then,CCK-8,microplate assay and Alizarin Red were used to detect the effects of miR-320 on proliferative activity,intracellular ALP protein activity and osteogenic ability.RT-q PCR was used to analyze the m RNA levels of osteogenesis-related genes(HOXA10,Runx2,BGP,COL-I,ALP).Results 1 AB-BMSCs were spindle under the microsco,positive for mesenchymal stem cell markers CD44 and CD90 and negative for hematopoietic stem cell marker CD34.These characteristics were in accord with BMSCs.2 Compared with the control group,the proliferation activity,intracellular ALP activity,calcium nodule formation in miR-320-overexpressed AB-BMSCs were significantly increased(P<0.01,P<0.01,P<0.01,respectively).And overexpression of miR-320 markedly decreased the m RNA levels of osteogenesis-related genes(HOXA10,Runx2,BGP,COL-I,ALP)in BMSCs(P<0.01,P<0.01,P<0.05,P<0.01,P<0.01,respectively).3 Compared with the control group,the calcium nodule formation ability of miR-320-knockdowned cells was significantly increased(P<0.01).Whereas,miR-320 knockdown did not affect the proliferation activity and intracellular ALP activity of AB-BMSCs(P>0.05,P>0.05,respectively).Compared with the control group,the m RNA levels of HOXA10,Runx2,BGP were significantly increased(P<0.01,P<0.01,P<0.01,respectively),while COL-I and ALP were no changes(P>0.05,P>0.05,respectively)in miR-320-knockdowned AB-BMSCs.Conclusions 1 Human AB-BMSCs cultured in vitro were in accord with characteristics of BMSCs.2 miR-320 negatively regulates the proliferation and osteogenic differentiation abilities of AB-BMSCs,which partially due to that it negatively regulates ALP activity,mineralization ability and the expression of some osteogenesis-related genes.