The Key Regulation Of MiR-124-3p During Reprogramming Of Primary Mouse Hepatocytes Into Insulin Producing Cells

Objective: To transdifferentiate mouse hepatocytes into insulin producing cells(IPCs)by small molecule compounds,to establish a therapeutic treatment for reprogramming primary hepatocytes into IPCs by non-transgenic method and to investigate the role of miRNA-124-3p in the differentiation of mouse hepatocytes into IPCs by small molecule compounds.Methods: Mouse primary hepatocytes were isolated,cultured and identified.Hepatocytes were transdifferentiated into IPCs by small molecule compounds,and detect the markers of each stage during the differentiation by qPCR and immunofluorescence.Insulin release experiments was used to assay the function of IPCs.miRNA-124-3p mimic and inhibitor were designed and transfected into mouse hepatocytes,respectively,then the change of expression of miRNA-124-3p and the target gene Foxa2 through qPCR were to determine whether the vector was successfully constructed,.The miR-124-3p up-regulated and down-regulated hepatocytes were induced to differentiate into IPCs.The expression of insulin-related genes and protein expression in each stage was detected by qRT-PCR and immunofluorescence.Mouse insulin enzyme-linked immunosorbent assay(ELISA)was used to detect the difference of insulin secretion of each stage during differentiation.Results: 1.The glycogen staining showed that the isolated cells were hepatocytes containing glycogen particles,which were further confirmed by immunofluorescence staining.The β-cell function-related genes of GK,Glut2 and insulin1 were expressed in IPCs.Immunofluorescence showed that insulin and C-peptide protein were positive,and the IPCs can secreted insulin under high glucose stimulation.Insulin secretion assay demonstrated the IPCs were functional.2.After transfected with mimic,the expression of miR-124-3p was significantly up-regulated,and the expression level of target gene Foxa2 was down-regulated compared with the control group.After transfection with miR-124-3p inhibitor,the expression level of target gene Foxa2 was up-regulated,and the difference was statistically significant.The miR-124-3p overexpression and inhibition vector construction were successful.The expression of Foxa2,PDX1,NeuroD,insulin1 and insulin2 in IPCs in the miR-124-3p inhibition expression group were significantly up-regulated,while the results were opposite in the miR-124-3p overexpression group.The results of cell immunofluorescence and glucose stimulation in vitro of miR-124-3p inhibition expression group showed that the expression of insulin,pdx1 and C-peptide was increased and the differentiation efficiency was higher than that of control group and overexpression group.Conclusion: 1.The primary mouse hepatocytes were successfully reprogrammed into insulin producing cells by small molecule compounds.2.miR-124-3p plays a negative regulatory role in the differentiation of hepatocytes into IPCs in vitro.Inhibition of miR-124-3p expression significantly increased the expression of FOXA2 and PDX1,promoted the differentiation of hepatocytes into IPCs,and increased the induction efficiency.Overexpression of miR-124-3p significantly inhibited the expression of FOXA2 and PDX1,and decreased the induction efficiency.