The Role Of MiR-152-3p In Regulating Human Induced Pluripotent Stem Cells Differentiation Into Insulin-producing Cells

Objective:Our previous study found that mi R-152-3p gradually increased in the process of stem cell differentiation into insulin producing cells,but its potential role and mechanism are not yet clear.Therefore,this study mainly focused on the role of miR-152-3p in the process of differentiation human induced pluripotent stem cells into insulin producing cells,and whether it can promote the efficiency and maturity of insulin producing cellls differentiation,will lay the foundation for the effective treatment of diabetes by stem cells in the future.Methods:The differentiation of human induced pluripotent stem cells into insulin producing cells was induced by using the four-step method and cell morphological changes during cells differentiation were observed under inverted microscope.In order to construct human induced pluripotent stem cells lines with mi R-152-3p overexpression or inhibition expression,the human induced pluripotent stem cells were infected by lentiviral vector with miR-152-3p overexpression or inhibition expression.The differentiation of human induced pluripotent stem cells with miR-152-3p overexpression or inhibition expression was induced by using the four-step method.The definitive endoderm cells,pancreatic progenitor cells and insulin producing cells were collected and the landmark genes Sox17,Foxa2,PDX1,NKX6.1,Insulin,MAFA were detected by real-time PCR.The key transcription factors Sox17,Foxa2,PDX1,NKX6.1,Insulin were detected and located by immunofluorescence.The differentiation efficiency in each stage was detected by flow cytometry,and the response ability of insulin producing cells to glucose was detected by glucose stimulation test.Results:During the differentiation of human induced pluripotent stem cells into insulin producing cells,miR-152-3p was up-regulated and its fold change was approximately 2,10,70,200 in the stage of definitive endoderm cells,progenitor foregut cells,pancreatic progenitor cells and insulin producing cells respectively.MicroRNA-152-3p and miR-152-3p downstream target gene DNMT1 were up-regulated and their fold change were approximately 18 and 4 respectively,so the human induced pluripotent stem cell lines of miR-152-3p overexpression and its inhibition expression were constructed successfully.The expression levels of SOX17,Foxa2,PDX1 and NKX6.1 in miR-152-3p inhibition expression cell line were more significantly up-regulated than in control cells,While those in miR-152-3p overexpression cell line were more significantly down-regulated than in control cells.The proportion of Sox17~+Foxa2~+or PDX1~+NKX6.1~+cells and insulin~+cells in miR-152-3p inhibition expression cell line was higher than that in control cells and miR-152-3poverpressioncellline(P<0.05),whilethoseinmiR-152-3p overexpression cell line were opposite than control cells(P<0.05).The differentiation efficiency of insulin producing cells in the control,miR-152-3p inhibition expression and overexpression group were 20.8%,28.5%,11.2%respectively and inhibition expression was higher than that of control or overexpression(P<0.05),while the miR-152-3p overexpression group was less than control(P<0.05).Glucose stimulation experiment showed that the secretion level of insulin and C-peptide were significantly increased in the miR-152-3p inhibition expression group than in control and miR-152-3p overexpression group(P<0.01),while the miR-152-3p overexpression group was lower than control(P<0.05).Conclusion:In this study,the human induced pluripotent stem cell lines with miR-152-3p overexpression or inhibition expression were constructed.The results show that the inhibition expression of miR-152-3p can promote the differentiation of human induced pluripotent stem cells into insulin producing cells demonstrating the expression of mark genes and key trancription factors at the various stages of differentiation,and enhanced the differentiation efficiency and insulin synthesis and secretion ability of insulin producing cells.However,the overexpression of miR-152-3p can inhibit the differentiation of human induced pluripotent stem cells into insulin producing cells and the synthesis and secretion of insulin.It is indicated that miR-152-3p plays a negative role during the differentiation of human induced pluripotent stem cells into insulin producing cells in vitro.