| [Background]Diabetes mellitus is a complex disease characterized by absolute insulin deficiency or resistance leading to hyperglycemia.Two major forms of diabetes exist,namely type 1 diabetes mellitus(T1DM)and type 2 diabetes mellitus(T2DM).T1 DM mainly appears in children.T1 DM occurs when the insulin-producing ?-cells in the pancreas are destroyed,resulting in insufficient insulin production.The etiology and pathogenesis of diabetes is very complex,it's not fully clarified so far.And it brings both great physical and psychological trauma to children who need taking insulin injections for the whole lifetime.So to explore the molecular mechanisms related with the pathogenesis of type 1 diabete and the interventions has become a top priority in the filed of pediatric endocrine research.The occurrence of T1 DM maybe related to the abnormal apoptosis of islet beta cells caused by some reasons.Researchs have shown that pro-inflammatory cytokines are mediators of apoptosis of islet beta cells in T1 DM.Recent observations indicate that ER stress signaling participates in maintaining the ER homeostasis of pancreatic ?-cells.It has been suggested that the reactive apoptosis pathway of ER stress has important roles in ?-cell death.This find illustrates the process of T1 DM from a new perspective.The early steps of insulin biosynthesis occur in the ER,and the ?-cells has a highly developed and active ER.It's one of the most sensitive cells to ERS.Persistent ERS and defective ERS signaling pathways in ?-cells can cause the dysregulation of ER homeostasis,which results in ?-cell apoptosis and autoimmune response,and then developing into diabetes mellitus.Since recent findings indicate that miRNAs play an important role in pancreas development,pancreatic ?-cell functions and the development of diabetes.MicroRNAs(miRNAs)are endogenous short singlestranded non-protein-coding RNAs with about 20~25 nucleotides in length which play a key role in post-transcriptional regulation of gene expression by partially complementary binding to the 3' untranslated region(3'UTR)of target mRNAs.In most cases,miRNAs play an inhibitory role in gene expression through downregulating the transcription or degrading the target mRNA to reduce the expression level of target protein.Then they participate in the regulation of cell growth,apoptosis,differentiation,hormones secretion and many other physiological processes and diseases.Our laboratory groups have found that miR-29 a highly expresses in the pancreatic tissue of the Intrauterine Growth Retardated(IUGR)newborn rats by screening with the technology of miRCURY TM LNA Array.Further bioinformatic analysis found that Mcl-1 maybe one of the target genes of miR-29 a.Mcl-1 is an anti-apoptotic Bcl-2 family member.Many recent observations suggest that Mcl-1 is the critical factor of cell apoptosis and survival in many tissues and cell lines.It takes part in regulating ERS.So it is speculated that Mcl-1 maybe an important factor connecting ERS and apoptosis.Our research will explore the effects of Mcl-1 on the apoptosis of pancreatic ?-cell and ERS through changing the expression levels of miR-29 a and Mcl-1.This research respectively expounds effects of overexpression or downregulation of rno-miR-29 a or Mcl-1 on the ERS and apoptosis in INS-1.We analyze the mechanism of INS-1 apoptosis mediated by miR-29 a.We will verify whether Mcl-1 is the target gene of miR-29 a in pancreatic ? cell line.Our findings suggest a new target and method to prevent and cure diabetes.ObjectivesTo study the mechanism underlying the regulation of ERS-induced apoptosis mediated by miR-29 a through regulating the expression of Mcl-1 in INS-1 cells.MethodsTransfecting INS-1 cells,which belong to pancreatic ? cell lines of rat,with miR-29 a mimics or inhibitors with the cDNA encoding Mcl-1(pcDNA3.1/Mcl-1)or small interference RNA(siRNA)of Mcl-1,then we detected the expression levels of miR-29 a,anti-apoptotic Bcl-2 family members Mcl-1 and bcl-2,ERS markers bip and chop with quantitative reverse transcripase-PCR(qRT-PCR)as well as Western Blot.The apptosis of INS-1 cells was analysised by the propidium iodide method using flow cytometry.According to the results of miRNA target gene prediction softwares analysis,the pGL3-promoter plasmid was modified by insertion of the Mcl-1-derived miR-29 a binding sites or a multi-base mutation into the 3'UTR.The pRL-SV40 vector was as the control vector.Then recombinant plasmid and pRL-SV40 vector were co-transfected with miR-29 a mimics into 293 T cells.293 T cells were chosen because they are readily transfected.The 293 T cells were harvested 48 h after transfection for dual luciferase analysis.ResultsOverexpressing miR-29 a promoted apoptosis and increased the proteins levels of bip and chop,two commonly used indicators of activation of ERS,while the antiapoptotic proteins Mcl-1 and bcl-2 were decreased markedly.In contrary,downregulating miR-29 a by antisense inhibitor reduced apoptosis in INS-1 cells,although the difference was not significant,decreased the protein expression levels of bip and chop,and increased the expression of Mcl-1 and bcl-2 significantly.But the mRNA levels of them all had no significant changes.Overexpressing miR-29 a and up-regulation of Mcl-1 by vectors overexpressing Mcl-1 could protect INS-1 cells against overexpressing miR-29 a induced apoptosis.The mRNA and protein expression levels of bip and chop both decreased and the protein expression of Mcl-1 and bcl-2 significantly increased.The expression level of Mcl-1 mRNA increased,while the bcl-2 mRNA had no significant changes.Inhibition of Mcl-1 by siRNA rendered INS-1 cells sensitive to apoptosis induced by overexpressing miR-29 a.Inhibition of Mcl-1,the expression level of Mcl-1 mRNA and protein decreased,promoted apoptosis and increased the proteins levels of bip and chop,the antiapoptotic proteins bcl-2 were decreased markedly,while its mRNA had no significant changes.Downexpression miR-29 a and upregulation of Mcl-1 by vectors overexpressing Mcl-1 could protect INS-1 cells rendered the protection of downexpressing miR-29 a.The protein expression levels of bip and chop decreased and the expression of Mcl-1 and bcl-2 significantly increased.Downexpression miR-29 a and inhibition of Mcl-1 by siRNA could destroy the protection of downexpressing miR-29 a,the apoptosis of INS-1 increased.The proteins levels of bip and chop increased,while the antiapoptotic proteins Mcl-1 and bcl-2 were decreased markedly.Inhibition of Mcl-1,the expression level of Mcl-1 mRNA decreased,but the mRNA level of bcl-2 had no significant change.After we co-transfected the luciferase construct with the mir-29 a target site from Mcl-1 3'UTR into 293 T cells with miR-29 a mimics,we found that the expression of luciferase significantly decreased.This suppression is relieved by a multi-base mutation in the biding site.ConclusionMi R-29 a mediated ERS-induced apoptosis through directly regulating the expression of Mcl-1 by binding to its target sequence in INS-1 cells. |
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