| Mammalian pancreas is a mixed gland containing the exocrine portion and endocrine portion.The pancreatic islets secrete glucagon and Insulin,which regulate organic metabolism and glucose homeostasis of body.The abnormal structure and function of endocrine ? cells could induce diabetes.The exocrine cells secrete digestive enzyme to digest food,while dysfunction of exocrine cells leads to metabolic defect.Deep researches of the formation mechanism of pancreatic tissue and the regulation of cell function can help to better understand the causes of pancreatic diseases,thus providing a basis for diagnosis and treatment of pancreatic diseases.Pancreatic blastema originates from early endoderm,it develops into pancreas through pancreatic progenitor cell proliferation,branch formation,exocrine and endocrine cell differentiation and cell migration.The proliferation and differentiation of pancreatic progenitor cells are regulated by transcription factors,such as Ptfla and Ngn3,and signaling pathways.microRNAs(miRNAs)play an important role in the individual development.The primary transcript of miR-17-92 cluster is processed into six miRNAs,miR-17,miR-20a,miR-18a,miR-19a,miR-19b and miR-92a.In recent years,the functions of miR-17-92 cluster on the spermatogenesis,neurogenesis,angiogenesis,and alveoli development have been revealed.However,there is little understanding of the regulation of pancrease by miR-17-92 cluster.This study explored the function of miR-17-92 cluster on the pancreatic progenitor cells,endocrine and exocrine cells.The results are showed as follows:1.Regulation of miR-17-92 cluster on the pancreatic progenitor cells.The family members were transfected into pancreatic progenitor cells.The research results of cell counting,CCK8 assay,and tissue section immunofluorescence staining showed that miR-18a inhibited the proliferation of pancreatic progenitor cells and pancreatic explants,and miR-19b increased the number of cells slightly.EdU,flow cytometry and qPCR results showed that miR-18a arrested the cell cycle at G1 period,while miR-18a inhibitor promoted pancreatic progenitor cells entrance into S stage.QPCR and Western results showed that miR-18a inhibits cell proliferation by targeting the expressions of CTGF and Nedd9,further counteracting the activation of AKT and ERK signaling,inhibiting the expression of CyclinDs,and promoting P21 and P27 expression.Results of cell counting,CCK8 and flow cytometry indicated that miR-19b promoted pancreatic progenitor cells entrance into S stage,and reversed the inhibitory function of miR-18a on the pancreatic progenitor cell proliferation partially,suggesting there is antagonism between miR-18a and miR-19b.QPCR for marked genes of the differentiated pancreatic progenitor cells confirmed that miR-18a promoted the expression of Ngn3 gene,the key transcription factor of endocrine cell differentiation,and miR-17,miR-20a induced the expression of exocrine gene Ptfla,amylase and elastase.2.Regulation of miR-17-92 cluster on the acinar cells.The bioinformatics prediction,dual-luciferase assay,qPCR and Western results showed that miR-17,miR-20a and miR-18a targeted the exocrine transcription factor Ptfla.miR-17 and miR-20a promoted Ptfla expression at transcriptional level,whereas miR-18a inhibited the expression of Ptfl a.miR-17IN,miR-20aIN and miR-18aTN suppressed the regulatory function of endogenous miRNAs on the Ptfla,To explore the regulation mechansim of miR-17 and mR-20a on the Ptfla gene,the luciferase activity of luciferase reporter vector with signal-nucleotide deletion and truncated Ptfla 3'UTR was analysed by dual luciferase report experiments,and the results showed that the regulation of miR-17 and miR-20a on the Ptfla gene depended on signal-nucleotide bulge in target site and flanking sequences outside target site.Results of qPCR,Western and ELISA showed that miR-17,miR-20a and miR-18a affected the expression of exocrine genes and the synthesis of digestive enzyme by targeting Ptfla.miR-17 and miR-20a promoted the expression of amylase and CPAI genes and amylase synthesis,however,miR-18a inhibited the expression of amylase and CPAI genes and the synthesis of amylase.ELISA results indicated that there was antagonism between miR-17/miR-20a and miR-18a,miR-17/miR-20a partially reversed the inhibitory function of miR-18a on the amylase synthesis.3.Regulation of miR-17-92 on the ? cells.The bioinformatics prediction,dual-luciferase assay,qPCR and Western results showed that Ngn3,the key transcription factor of endocrine cell differentiation,was the target gene of miR-17,miR-20a and miR-18a.miR-17 and miR-20a inhibited Ngn3 expression,but miR-18a promoted the expression of Ngn3.To explore the regulation mechansim of miR-18a on the Ngn3 gene,the luciferase activity of luciferase reporter vector with signal-nucleotide insertion and truncated Ngn3 3'UTR was analysed by dual luciferase report experiments,and the results showed that the regulation of miR-18a on the Ngn3 gene depended on the combination between seed sequences and target site,and the flanking sequences CUUCUU.Results of qPCR,Western,ELISA showed that miR-18a enhanced insulin synthesis in the P cells,whereas miR-17 and miR-20a slightly inhibited insulin synthesis.miR-17IN,miR-20aIN and miR-18aIN suppressed the regulatory function of endogenous miRNAs on Insulin gene.Conclusion:1.Members of miR-17-92 cluster regulated the proliferation and differentiation of pancreatic progenitor cells.miR-18a inhibited the proliferation of adult pancreatic progenitor cells by targeting the gene expressions of connective tissue growth factor(CTGF),and neural precursor cell expressed,developmentally down-regulated 9(Nedd9),as well as by suppressing activation of the proliferation-related signaling pathways phosphatidylinositol 3-kinase-protein kinase B(PI3K/AKT)and extracellular signal-regulated kinase(ERK).miR-18a arrested the cell cycle at G1 stage,whereas miR-19b promoted pancreatic progenitor cell entrance into S stage.There was antagonism between miR-18a and miR-19b,and miR-19b reversed the inhibitory function of miR-18a on the pancreatic progenitor cell proliferation partially.miR-18a promoted the expression of Ngn3 gene which is the key gene of endocrine cell diflferentiation.miR-17,miR-20a induced Ptfla expression,which is the key gene of exocrine cell differentiation,and promoted the expression of exocrine genes amyalse and elastase.2.miR-17-92 cluster regulated the exocrine transcription factor Ptfla and amylase synthesis.miR-17 and miR-20a promoted Ptfla expression and amylase synthesis,and miR-18a inhibited the expression of Ptfla and amylase synthesis.The regulation of miR-17 and miR-20a on the Ptfla gene depended on signal-nucleotide bulge in target site and flanking sequences outside target site.There was antagonism between miR-17/miR-20a and miR-18a,miR-17/miR-20a partially reversed the inhibitory function of miR-18a on the amylase synthesis.3.miR-17-92 cluster regulated Ngn3 gene,the key transcription factor of endocrine cell differentiation,and Insulin synthesis in the ? cells.miR-17 and miR-20a inhibited the expression of Ngn3 gene,but miR-18a promoted Ngn3 expression.The regulation of miR-18a on the Ngn3 gene depended on the combination between seed sequences and target site and flanking sequences CUUCUU.miR-18a enhanced insulin synthesis in the ? cells,whereas miR-17 and miR-20a slightly inhibited insulin synthesis. |
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