The Relationship Between MiR-375 And The Differentiation Of The Human Embryonic Stem Cells Into Insulin-producing Cells

Human embryonic stem cells(hESCs)are pluripotent stem cells,which has the capacity of infinite replication,self-renew and differentiating into various cell types cultured in vitro.HESCs can differentiate into insuling-producing cells(IPCs)cultured in certain medium,which can be transplanted to treat type ? diabates to compensate the shortage of organ resources.MiR-375 is an essential transcriptional regulation factor in the process of pancreatic development and maturation,which is expressed in both ?-cells and non-?-cells and promotes transcription of insulin and proliferation of(3 cells.Therefore,it is of great significance to research the function of miR-375 in the differentiation of hESCs into IPCs for promoting the efficiency of differentiation and obtaining the mature(3 cells that only express insulin.In present study,the hESCs were cultured in various media to differentiate into IPCs,and the differentiation included four stages:definitive endoderm(Stage ?),primitive gut(Stage ?),endocrine precursor(Stage ?)and pancreatic endocrine(Stage ?).Retinoic acid(RA)was addad in differentiation culture medium at various stages.Overexpression of miR-375 of hESCs was mediated by lentivirus and liposome respectively.In order to invesgate the influence of overexpression of miR-375 to hESCs,the expression level of miR-375 and genes related to pancreatic differentiation incluing Foxa2,Cxcr4,Ngn3,Pdx1,Insulin,Glucagon and Somatostatin were examined by qRT-PCR.Synthesis and secretion of insulin were examined by immunofluorescence assay and elisa.1.To obtain the most appropriate differentiation culture process,2 ?M RA was added to the medium at the different period of definitive endoderm into endocrine precursor.The groups included RA was not added to the medium;RA was added to the medium at Stage II;RA was added to the medium at Stage ?;RA was added to the medium at both Stage II and Stage III.We found that hESCs could not differentiate into IPCs without RA.Foxa2,Cxcr4,Ngn3,Pdxl,Insulin,Glucagon,Somatostatin highly expressed under the condition of RA added to the medium at Stage II,and in which growth of the cells was healthy.Therefore,the most appropriate differentiation condition was to add 2 ?M RA to the medium at Stage ?.2.Overexpression of miR-375 mediated by lentivirus made a great influence on the state of the cells.The capacity of proliferation and differentiation was decreased and the cell apoptosis was increased.A large number of cells died at the early stage of differention,which led to the failure of differentiation of hESCs into IPCs.3.HESCs transfected with miR-375 mimic mediated by liposome made a great influence on differentiation.Overexpression of miR-375 at the early stage of differentiation increased the expression of Foxa2 and Cxcr4,at the stage ? increased the expression of Ngn3 and Pdxl,and at the end of differentiation increased the expression of Insulin and reduced the expression of Glucagon and Somatostatin at the same time.Besides,overexpression of miR-375 promoted synthesis and secretion of insulin.Therefore,overexpressing of miR-375 in the differentiation of hESCs into IPCs increased the expression level of genes related to development and maturation of P cells,and reduced the expression of other hormone genes except insulin.Meanwhile,it promoted the mature of ? cells and increased insulin secretion.