Overexpression Of MiR-126Induces Apoptosis Of INS-1Cells

Background With changes in life styles and social environments, the incidence ofdiabetes mellitus (DM) and other metabolic diseases is increasing continuously. DM iscurrently considered to be characterized by decreased sensitivity of peripheral tissues andorgans to insulin and dysfunction of β cells in secreting insulin. Recent studies havedemonstrated that insulin synthesis and secretion can be regulated and promoted by selffeedback of β cells. In addition, aging of β cells can also influence insulin secretion,contributing to the pathogenesis of DM2. It is reported that there exist miR-126target genesin signaling pathways of insulin, but the association between miR-126and apoptosis andproliferative pathogenesis of INS-1cells remains unclear. This study was attempted to explorethe possible mechanism underlying the regulatory effect of miR-126on INS-1cells.Methods INS-1cells were transfected with miR-126mimics or miR-126inhibitor for48h and then stimulated with100nM insulin for12h. The experiment was performed in fourgroups: group A, negative control (NC) group; group B, miR-126overexpression group(transfected with miR-126mimics); group C, miR-126low expression group (transfected withmiR-126inhibitor); and group D, positive control group (transfected with miR-375mimics).Apoptosis and vitality of INS-1cells were detected by flow cytometry using Annexin V/PIdouble-stained method and MTT respectively. XIAP protein expression was detected byWestern blot.Results1) To determine the regulatory effect of miRNA expression on INS-1cells,miR-126mimics or inhibitor was transfected into INS-1cells, and miRNA expression levelwas measured by quantitative PCR. The results showed that artificial synthetic miRNA effectively regulated miRNA expression in vitro (221.45±23.08vs.10.86±2.52).2) Theapoptosis rate of INS-1cells was detected by flow cytometry. It was found that the number ofapoptotic cells was increased in overexpression group (93.59±1.11vs.74.72±7.35; P<0.05),and that the viability of INS-1cells was decreased as shown by MTS assay (16.216±0.879vs.31.842±0.912; P<0.05).3) Cell ultrastructure was observed by electron microscopy.Compared with NC and downexpression groups, the number of necrotic cells was increased inover-expression group; the cell size became smaller; more nuclei became creased or rippled;chromatin was concentrated and peripherally aggregated; mitochondria underwent vacuolarchanges; the endoplasmic reticulum was expanded and fused with the cell membrane,showing a foaming phenomenon.4) Omi/HtrA2and caspase-3were mainly expressed in thecytoplasm, and XIAP was expressed in the nucleus. Analysis of cell fluorescence intensity ofthe proteins in INS-1cells showed that the expression levels of the three proteins wereincreased slightly in overexpression group as compared with low expression group. WesternBlot showed that XIAP was downregulated in overexpression group when INS-1cells werestimulated with100nM insulin for48h after transfection with miR-126mimics.Conclusion1. miR-126over-expression could induce apoptosis and inhibitproliferation of INS-1cells.2. Over-expression of miR-126and miR-375could decrease XIAPexpression of INS-1cells and induce apoptosis.