| Background:Diabetes is considered to be one of the common chronic diseases characterized by disordered glucose metabolism. Many findings suggest that glucose metabolism is associated with metabolic memory of hyperglycemia,inflammation and mitochondrial dysfunction, however,the exact mechanism remains unclear.Micro RNAs are a large class of evolutionarily conservative,non-protein-coding, small RNA molecules of 20-25 nucleotides in length. After mi RNAs bind to the 3’ untranslated region(UTR) of their target gene through principle of complementary base pairing, they can regulate gene expression at the post-transcriptional leve by inhibiting translation of m RNA or degrading m RNA.mi RNAs participate in the regulation of pathphysiological process just because of the function of mi RNA. A large number of experimental data suggests that there is a close correlation between mi RNAs and the development of diabetes and the maintainance of glucose homeostasis. Anna Zampetaki et al found that plasma micro RNA126 remarkably decreased in patient with T2 DM,and it may be a new marker for risk estimation to distinguish between normal group and patient with T2 DM. In 2013,our research highlighted the presence of mi R-126 was signficantly low in patients with T1 DM and T2 DM. Beside,the expression level of it was closely associated with risk factors and complication of DM, for example,inflammation,lipid metabolism and so on.The liver,which is the most important place for the progress of glucose metabolism,plays a crucial role.Using overexpression of mi R-126, we explor whether mi R-126 impacts the glucose metabolism and responsiveness to insulin in normal human liver cells. The present study may provide a novel insight into the pathogenesis of insulin resistance and DM. Methods:1.Normal human liver cells were cultured in vitro. Cells were divided into three groups: NC-FAM group, inhibitor NC-FAM group and normal control group.Cells of each group was transferred with respective sequence at different concentrations of 10, 20, 40 and 80 n M via lip2000, then fluorescence microscopy was used to observe transfection efficiency in order to determine the optimal transfection conditions.2.Cells were divided into 6 groups : mi R-126 mimics group, mi R-126 inhibitor group, Negative Control group, micro RNA inhibitor NC group, lip2000 group and normal control cell group,and every group included 6 samples. Under the most effective transfection conditions ascertained above, the artificial synthetic mi R-126 mimic, mi R-126 inhibitor, Negative Control and micro RNA inhibitor NC were transferred via lip2000.3.The transfection efficiency was tested by quantitative real-time PCR(q RTPCR).4.Cells were continued cultured for 48 h and then stimulated with synthetic insulin(100n M).GOD-POD method, BCA protein assay kit, anthrone method, lactate assay kit and pyruvate kinase kit were respectively used to observe glucose concentration in the supernatant, the protein concentration of the lower cell of plates, glycogen content,lactic production and pyruvate kinase activity. The ratio of glycogen to protein content(μmol·mg-1) indicated the account of glycogen synthesis; under the action of lactic acid, the ratio of the glucose concentration in the supernatant to protein concentration(mmol · mg-1) represented the account of gluconeogenesis; under the high glucose level, the ratio of lactic acid concentration to protein concentration(mmol · mg-1) and pyruvate kinase activity to protein concentration(U · gprot-1) indicated the account of glycolysis. Through these indicators, the effect of mi R-126 at different concentrations on hepatic glucose metabolism was observed. Result:1.On a serum-free condition without antibiotics, six hours after transfection, observed by fluorescence microscopy, NC-FAM and inhibitor NC-FAM were effectively transfected into normal human liver cells in a dose-dependent manner via lip2000.And the appropriate concentration of nucleotide was 80 nm.2.Mi R-126 expression level was measured by q RT-PCR, the results showed that the level of mi R-126 of the mi R-126 mimic transfection group is significantly higher than the other groups, and the difference is statistically significant(P <0.05).3.Using the most effective condition ascertained above, the results showed that the glucose utilization significantly decreased(33.66±2.55 vs 59.12±4.03,P <0.05),the indicator of glycogen synthesis reduced(4.35±0.33 vs 8.42±0.56,P <0.05), the account of lactate production reduced(12.28±1.72 vs 22.51±1.25,P <0.05),and the pyruvate kinase activity significant decreased(120.87±26.23 vs 277.39±57.60,P <0.05) in mi R-126 mimic group.Besides, the results showed that the account of gluconeogenesis of mi R-126 mimic group effectively increased(17.05±0.88 vs 13.05±0.41,P<0.05) and the account of gluconeogenesis of mi R-126 inhibitor group decreased(7.41±1.39 vs 13.05±0.41,P<0.05). Conclusions:When mi R-126 expression level was increased in normal human liver cells, mi R-126 regulated glucose metabolism through decreasing glycogen synthesis, lactate production and pyruvate kinase activity, and increasing the amount of gluconeogenesis. The research showed that the over-expressing mi R-126 in hepatocytes reduced glucose utilization and sensitivity to insulin, and enhanced insulin resistance in hepatocytes. |
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