The MicroRNA Profile Of Human Pancreatic Islets And The Mechanism Of Mir-148 Regulation Of Pancreatic Beta Cell Proliferation And Apoptosis

Recent advances in the understanding of the genetics of type 2 diabetes(T2D)susceptibility have focused on the regulation of transcriptional activity within the pancreatic beta-cell.Emerging evidences suggest that microRNAs play an important role in insulin production,secretion and actions,as well as in pancreatic development and adult? cell physiology.The identification of tissue-specific microRNAs implicated in human pancreatic islets might be useful for the future development of effective strategies for early diagnosis and therapeutic intervention of type 2 diabetes.We set out to establish the microRNA profile of human pancreatic islets and to explore their potential involvement in T2D susceptibility.In this study,we isolated human primary islets through perfusion digestion and continuous gradient centrifugation,extracted human islets total RNA,and used Illumina small RNA sequencing to profile the microRNA fraction of primary human islets;Validated the sequencing results by real-time qPCR chip technology;miR-148 highly expressed in human islets,To explore the mechanism of miR-148 regulation of pancreatic beta cell proliferation and apoptosis,MIN6 cell line and primary islet dissociated cells were transfected with inhibitor of control or miR-148,in the indicated time period post transfection,we detected cell cycle by immunostainning and FACS,and detected cell apoptosis by FACS and TUNEL;Then,by using real-time qPCR and western-blot,screened target genes of miR-148.Studied effects of miR-148 knockdown on the target genes 3'UTR luciferase in beta-cells.We used Illumina small RNA sequencing to profile the microRNA fraction of primary human islets,In total,more than one thousand kinds of microRNAs were found to be expressed in human islets,miR-148 is highly expressed microRNAs in human islets.In mouse primary islet ?cells and ?cell line,knockdown of miR-148 inhibited cell growth,and promoted cell apoptosis.Computer-aided algorrithms predicted pten,bim,and p27 as the potential target gene of miR-148.Indeed,miR-148 knockdown increased the expression of them.Using luciferase reporter assays,we identified pten,bim,and p27 as the direct target genes of miR-148.Our findings suggest that miR-148 play important roles as a regulator of beta-cell proliferation.