The Role Of MiR-124 And MiR-375 In The Proliferation Of Pancreatic Progenitor Cells

The pancreas is a mixed gland with both exocrine and endocrine portions.The exocrine acini secrete digestive enzymes into pancreatic ducts,which drain into the duodenum to digest food.Endocrine islet is comprised of α,β,PP,ε and δ cells,which control multiple homeostatic functions.Cell lineage tracing experiments have shown that all exocrine and endocrine lineage cells are generated from a common pool of multipotential progenitor cells during early pancreatic development.A few reports have shown a small population of pancreatic progenitor cells with high proliferative capability and multipotential remains in adult pancreata,which function in pancreatic growth and tissue maintenance.microRNAs(miRNAs)are a class of small,non-coding RNAs that regulate gene expression.The base pairing interactions between miRNAs and their target mRNAs,often within the 3’-untranslated region(UTR)of target genes,result in the degradation of target mRNAs or repression of their translation.In recent years,several studies have shown that miRNAs contribute to pancreatic development and function maintenance.In Dicer(an essential enzyme for miRNAs formation)null mutation mice,the blockage of mature miRNAs formation led to gross defects in all pancreatic lineages,and the differentiation of endocrine cells especially p-cells was inhibited.miR-375 was found to be involved in regulating insulin secretion and maintaining α and β cell mass.miR-124 could influence insulin secretion through repressing Foxa2 expression in β cell.However,the function of miR-375 and miR-124 in the proliferation or differentiation of pancreatic progenitor cells is largely unknown.In this study,the expression of miR-375 and miR-124 in early mouse embryonic pancreas and adult pancreatic cell lines were firstly detected.And then whether miR-375 and miR-124 could participate in the proliferation of pancreatic progenitor cells was analyzed.Finally,whether overexpression miR-375 and miR-124 could promoting pancreatic progenitor cells differentiating into endocrine cells was detected.We found that miR-375 and miR-124 overexpression could inhibit the proliferation of pancreatic progenitor cells.Using bioinformatics predicting software Targetscan and experiments of cell biology,we screened and identified the target genes of miR-375 and miR-124.Our research will deepen understanding of molecular mechanism of pancreatic progenitor cell proliferation and pancreatic development.Our study may provide theoretical basis for the treatment of pancreatic diseases such as diabetes.1.Materials and Methods1.1 The expression of miR-375 and miR-124 in early mouse embryonic pancreas and adult pancreatic cell lines.The total RNA of E12.5 and E15.5 mouse embryonic pancreas,pancreatic progenitor cells and β cell line MIN6 were extracted.The total RNA was added polyA tail and then was reverse transcribed into cDNA.The expression of miR-375 and miR-124 was determined by RT-PCR and qRT-PCR.1.2 The effects of miR-375 and miR-124 overexpression on pancreatic progenitor cells proliferation.Pancreatic progenitor cells were transfected with miR-375 and miR-124 mimics.72h after transfection,cells proliferation was evaluated by blood count plate method,CCK-8 assay and Ki67 immunostaining assay.13 The effects of suppressing miR-124 on pancreatic progenitor cells proliferation.Pancreatic progenitor cells were transfected with miR-124 inhibitor(2’-O-Me modified antisense oligonucleotides).72h after transfection,cells proliferation was evaluated by CCK-8 assay.1.4 Prediction and validation of miR-375 and miR-124 targets.Overexpression of miR-375 and miR-124 inhibited pancreatic progenitor cells proliferation.By using the algorithms Targetscan,we considered CCND2,STAT3,IQGAP1,E2F3,EPS8,GRB2,PIK3CA and SOS1 as putative targets of miR-124;YAP1 as a putative target of miR-375.Dual luciferase reporter assay and westen blot analysis were generated to validate whether miR-124 could inhibit CCND2,STAT3,IQGAP1,E2F3,EPS8,GRB2,PIK3CA and SOS1 expression;whether miR-375 could inhibit YAP1 expression.When these putative targets were knocked down by siRNA,cells proliferation was evaluated by CCK-8 assay and Ki67 immunostaining assay.1.5 The effects of miR-124 on PI3K/AKT and ERK1/2 signal pathway.Pancreatic progenitor cells were treated with 20μM LY294002(PI3K inhibitor),20μM U0126(MEK1/2 inhibitor)or DMSO.48h after treatment,phosphorylation of AKT and ERK1/2 were determined by western blot.Cells proliferation was evaluated by CCK-8 assay.Pancreatic progenitor cells were transfected with miR-124.72h after transfection,phosphorylation of AKT and ERK1/2 were determined by western blot.1.6 Whether miR-375 and miR-124 overexpression promote pancreatic progenitor cells differentiating into endocrine cells.Pancreatic progenitor cells were transfected with miR-375 and miR-124.7d after transfection,the pancreatic progenitor specific cells markers and endocrine cells specific markers was detected by RT-PCR.1.7 Whether miR-124 overexpression promotes pancreatic progenitor cells differentiating into nerve cell.Pancreatic progenitor cells were transfected with miR-124.7d after transfection,the pancreatic progenitor specific cells markers and nerve cell specific markers was detected by RT-PCR.2.Results2.1 The expression level of miR-375 and miR-124 in E12.5 mouse pancreas was low,and their expression were increased in E15.5 mouse pancreas.miR-375 was expressed highly in MIN6 cells,but was undetectable in pancreatic progenitor cells.miR-124 was expressed highly in MIN6 cells,but lower expression in pancreatic progenitor cells.2.2 When miR-124 and miR-375 overexpression in pancreatic progenitor cells,CCK8 assay and Ki67 immunostaining assay showed that cell proliferation activity was reduced.However,when endogenous miR-124 was inhibited in pancreatic progenitor cells,CCK8 assay showed that cell proliferation activity was not influenced.23 IQGAP1,SOS1,STAT3 and CCND2 were identified as miR-124 target genes by dual luciferase reporter assay and western blot analysis.RT-PCR assay showed that overexpression of miR-124 reduced IQGAP1 expression through mRNA cleavage,whereas miR-124 just inhibited SOS1,STAT3 and CCND2 protein translation,but no mRNA cleavage.Furthermore,using siRNA interferencing IQGAP1,STAT3 and CCND2 expression in pancreatic progenitor cells,reduced cell proliferation activity.However,interferencing SOS 1 didn’t influence cell proliferation activity.2.4 YAP 1 was identified as a miR-375 target gene by dual luciferase reporter assay and western blot analysis.RT-PCR assay showed that miR-375 overexpression reduced YAP1 expression through mRNA cleavage.In addition,pancreatic progenitor cell proliferation activity was reduced when YAP1 expression was knockdowned by siRNA.2.5 When miR-124 overexpression in pancreatic progenitor cells,western blot assay showed that phosphorylation of AKT(T308,S473)and ERK1/2 were reduced.When pancreatic progenitor cells were treated with LY294002,phosphorylation of AKT(T308,S473)were reduced and cells proliferation was inhibited.Furthermore,when pancreatic progenitor cells were treated with U0126,phosphorylation of ERK1/2 was inhibited,but cells proliferation was not influenced.2.6 When miR-124 and miR-375 overexpression in pancreatic progenitor cells,RT-PCRassay showed that the expression of pancreatic progenitor cell specific markers,such as Nestin,SOX9,Hesl and c-Met were no significant change.Furthermore,endocrine cells specific markers didn’t express when miR-124 and miR-375 overexpression.2.7 When miR-124 overexpression in pancreatic progenitor cells,cell appeared nerve synapse structure.RT-PCR assay showed that astrocyte cell specific marker GFAP appeared;The expression of tubb3,which was primarily expressed in neurons,was increased;However,mature neurons specific markers,such as MAP2 didn’t express.Conclusion3.1 Overexpression of miR-124 inhibited pancreatic progenitor cells proliferation though targeting IQGAP1,STAT3 and CCND2.Furthermore,miR-124 reduced pancreatic progenitor cells proliferation activity through inhibited PI3K/AKT signal pathway.When endogenous miR-124 was inhibited,pancreatic progenitor cell proliferation activity was not influenced,this may cause by the low level of miR-124 in pancreatic progenitor cells.3.2 miR-375 was undetectable in pancreatic progenitor cells.Ectopic expression of miR-375 inhibited pancreatic progenitor cells proliferation by targeting YAP1.3.3 Overexpression of miR-124 and miR-375 didn’t promote pancreatic progenitor cells differentiating into endocrine cells.