| BackgroundDiabetes mellitus(diabetes mellitus, DM) is a common endocrine and metabolic disease,pathologically characterized by absolute or relative deficiency of insulin secretion, or insensitivity of the peripheral tissues to insulin, disturbance of glucose metabolism including disorder protein and fat metabolism. The incidence of DM is on the rise yearly worldwide.According to the WHO estimates, the global number of DM will reach 300 million in 2030. It is estimated that China now has 92.4 million diabetic adults, accounting for the largest population of DM in the world. More than 90% DM patients suffer from type 2diabetes(DM2), seriously affecting their health. The pathogenesis of DM is complex, insulin resistance plays an important role in the process of the occurrence of DM2,but the exact mechanism of insulin resistance remains unclear.Insulin, the only hypoglycemic hormone of the human body, is a protein hormone secreted by pancreatic beta cells. Insulin signaling pathway involves a number of physiologic processes, including the change of calcium concentration on insulin secretion. It is reported that there exist Mi RNA-126 targets in the insulin signaling pathway. The previous work in our laboratory has also confirmed that insulin receptor substrate 1(IRS-1) is the target. But whether mi R-126 affects insulin synthesis and release of pancreatic beta cells remains unclear.In the present study, we used an insulinoma B cells(INS-1) model to investigate on theimpact of Mi RNA-126 on the calcium channel current of INS-1 cells, calcium ion concentration and insulin secretion.Methods1. INS-1 cells were plated on culture plates.Different mi RNAs were transfected with40 n M in INS-1 cells after 24 h.The transfection time was 48 h, INS-1 cells were then stimulated with 100 n M synthetic insulin for 12 h. mi R-126 gene expression in INS-1 cells was detected by fluorescence quantitative PCR.2. Using confocal laser technology,changes in INS-1 intracellular calcium concentrations were detected in four groups: negative control(NC) group without transfection, mi R-126 m group transfected with mi R-126 mimics(mi R126 m group), mi R-126 i group transfected with mi R-126 inhibitor(mi R-126 i group), and the positive control group, transfected with mi R-375mimics(mi R-375 m group).3. Using the perforated patch clamp technique, INS-1 cell membrane calcium channel currents were detected in NC group and mi R-126 m group.4. Using ELISA and flow cytometry, changes in the amount of insulin and secretionmi tochondrial membrane potential were detected in NC group, mi R-126 m group, mi R-126 i group, and mi R-375 m group.Result1. Compared with NC group(107.26 ± 37.88), the expression level of mi R-126 m group was significantly increased(398.77 ± 53.72 vs., p<0.05), and significantly decreased in mi R-126 i group(20.26 ± 7.827, P <0.05).2. Detection of the INS-1 intracellular calcium concentration by confocal laser technology showed that the basis of glucose(glucose 5.6m M) stimulated the cells: compared with NCgroup, the calcium ion concentration in mi R-126 m group was significantly higher(P<0.05),and significantly lower in mi R-126 i group(P <0.05);high concentration of glucose(16.8m M) stimulated the cells.There was no difference in the intracellular calcium ion concentration bettween mi R-126 m group and NC group(P>0.05),It was significantly low in mi R-126 i group(P <0.05). Detection of INS-1 cell membrane calcium channel currents by the perforated patch clamp technique showed that calcium influx was increased in mi R-126 m group as compared with NC group(P <0.05).3. ELISA detection of insulin secretion showed that compared the NC group, basal insulin secretion in mi R-126 m group was increased, and decreased in mi R-126 i group significantly.Insulin secretion showed no significant difference in the high glucose concentration between mi R-126 m group and NC group(P>0.05), and it was significantly lower in mi R-126 i group(P<0.05). Flow cytometry for mitochondrial membrane potential showed that compared with NC group, apoptosis in mi R-126 m group was increased(P <0.05), and decreased in mi R-126 i group(P <0.05).Conclusion1. mi R-126 over-expression could increase the intracellular calcium ion concentration and calcium influx of INS-1 cells.2. Over-expression of mi R-126 could increase basal insulin secretion and induce apoptosis. |
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