MiR-320c Regulates The Proliferation And Differentiation Of Human Dental Pulp Stem Cells

Objectives This study aims to investigate the effect of mi R-320 c on the proliferation,osteogenic and adipogenic differentiation abilities of human dental pulp stem cells(h DPSCs),as well as on the expression of osteogenesis-related and adipogenesis-related genes.This study will provide a theoretical basis for the application of h DPSCs in regenerative medicine.Methods 1 The cells were isolated and cultured by method which combination of tissue block and mixed enzyme digestion.Then,the cells were identified by the flow cytometry method.2 The h DPSCs were transfected with mi R-320 c mimic and negative control(mi RNC)for 24 hours.Then,CCK-8 was used to detect the proliferation ability of h DPSCs in the two groups.RT-q PCR was used to analyze the transfection efficiency and the m RNA levels of OCN,Runx2 and PPAR-? in the two groups.Alizarin Red was used to detect the osteogenic differentiation ability of h DPSCs in the two groups.Oil Red O was used to detect the adipogenic differentiation ability of h DPSCs in the two groups.3 The h DPSCs were transfected with mi R-320 c inhibitor and micro RNA inhibitor NC(mi R-INNC)for 24 hours.Then,CCK-8 was used to detect the proliferation ability of h DPSCs in the two groups.RT-q PCR was used to analyze the transfection efficiency and the m RNA of OCN,Runx2 and PPAR-? in the two groups.Alizarin Red was used to detect the osteogenic differentiation ability of h DPSCs in the two groups.Oil Red O was used to detect the adipogenic differentiation ability of h DPSCs in the two groups.Results 1 Cells were identified as h DPSCs,which were positive for mesenchymal stem cell markers CD44 and Strol-1 and negative for hematopoietic stem cell marker CD34.2 mi R-320 c overexpression and knockdown were confirmed in h DPSCs,respectively(P<0.01).3 Compared with the mi R-NC group,the proliferation ability of mi R-320 coverexpressed h DPSCs was significantly decreased(P<0.01).On the other hand,compared with the mi R-INNC group,the proliferation ability of mi R-320c-knockdowned h DPSCs was significantly enhanced(P<0.01).The results showed that the mi R-320 c could negatively regulate the proliferation ability of h DPSCs.4 Result of calcium node alizarin red staining: compared with the mi R-NC group,the calcium nodule formation ability of mi R-320c-overexpressed h DPSCs was significantly reduced(P<0.05).Result of RT-q PCR: compared with the mi R-NC group,the m RNA levels of OCN and Runx2 were significantly decreased(P<0.01).On the other hand,compared with the mi R-INNC group,the calcium nodule formation of mi R-320c-knockdowned h DPSCs was significantly increased(P<0.01).Result of RT-q PCR: compared with the mi R-INNC group,the m RNA levels of OCN,Runx2 were significantly increased(P<0.01).The results showed that the mi R-320c could negatively regulate osteogenic differentiation ability of h DPSCs.5 Result of Oil Red O staining: compared with the mi R-NC group,the lipid droplets formation of mi R-320 coverexpressed h DPSCs was significantly increased(P<0.01).Result of RT-q PCR: compared with the mi R-NC group,the m RNA levels of PPAR-? was significantly increased(P<0.01).On the other hand,compared with the mi R-INNC group,the lipid droplets formation of mi R-320c-knockdowned h DPSCs was significantly decreased.Result of RT-q PCR: compared with the mi R-INNC group,the m RNA levels of PPAR-? was significantly decreased(P<0.01).The results showed that the mi R-320 c could promotely regulate adipogenic differentiation ability of h DPSCs.Conclusions 1 The cells were isolated and cultured by the combination method of tissue block and mixed enzyme digestion in vitro,which show characteristics of mesenchyma stem cells.2 mi R-320 c negatively regulates proliferation ability of h DPSCs.3 mi R-320 c may negatively regulates osteogenic differentiation ability of h DPSCs by negatively regulating of the m RNA levels of OCN and Runx2.4 mi R-320 c could positively regulate adipogenic differentiation ability of h DPSCs by positively regulating of the m RNA level of PPAR-?.