| BackgroundWith the development of living level and the alteration of life style, the incidence of metabolic disorders has increased during the last decades. In the development of Type 2 diabetes, insulin resistance, which runs through the whole process and plays an important role in Type 2 diabetes. So it can cause some diseases such as obesity, polycystic ovary syndrome, hyperlipemia and atherosclerosis. Liver is important in glucose metabolism. Insulin adjusts glucose metabolism through IRS/PI3K/Akt signaling pathway in liver. However, the pathogenesis of insulin resistance is still unclear.Micro RNAs are a class of non-coding RNAs which have a significant impact on homeostasis and the occurring of disease. mi RNAs can bind to the 3’ untranslated region(UTR) of their target gene to inhibit the transcription. Micro RNA-126 is highly enriched in endothelial cells and endothelial apoptosis body. Previous research have already found that the expression of Micro RNA-126 was decreased in the plasma of diabetes animal models and diabetic patients. So we infer that Micro RNA-126 has relation with the the development of diabetes. Previous research put forward that Micro RNAs might not only be indicators of early disease onset but are also responsible for the progression of disease, so Micro RNAs associated with diabetes can not only affect the progress of the disease,but also a early intervention target of disease. Whether Micro RNA-126 can cause insulin resistance and affect insulin signal transduction pathway is useful to reveal in this experiment. ObjectiveThis research takes Chang liver cells as object, the Chang liver cells were transfected with Micro RNA-126 mimics, Micro RNA-126 inhibitor and the negative control sequence respectively. The expression level of IRS-1、PIK3R2、Akt-2 and p-Akt-2 were detected. This study aims to investigate the effects of Micro RNA-126 on glucose metabolism in the human liver cells. Materials and methods1.Chang liver cells were cultured in RPMI-1640 with 10% fetal bovine serum(FBS) at 37 °C in a 5% CO2 incubator.2.Fluorescence microscope to determine the optimum concentration of transfection:Chang liver cells seeded at 5.0×105 cells/well of a 24-well plate and grown to an approximate 70% confluence. Different concentrations(20 nmol/L、40 nmol/L、80 nmol/L)of FAM-NC and inhibitor FAM-NC were transfected to cells with Lipofectamine2000. Then observe the green fluorescence in the cell under the fluorescence microscope after 6 h.3.The object was divided into six groups : Micro RNA-126 mimics group, Micro RNA-126 inhibitor group, Micro RNA-126 mimics negative control group, Micro RNA-126 inhibitor negative control group, Lipofectamine 2000 group and normal control group. Micro RNA-126 mimics、Micro RNA-126 inhibitor and the negative control sequence were transfected with Lipofectamine2000 respectively. The relative expression of Micro RNA-126 was detected by quantitative real-time polymerase chain reaction(q RT-PCR).4.The protein expression of insulin receptor substrate(IRS-1) 、phosphatidylinositol 3-kinase p85β(PIK3R2) 、 Protein Kinase B-2( Akt-2)、phosphorylated Akt-2(p-Akt-2) in different groups were detected by Western blot.5.SPSS 17.0 statistical software was applied for the date statistics. The date was expressed with the meant standard deviation. The significance of difference was analyzed using Student’s t test for unpaired data and use one-way ANOVA for comparing among many groups. A value of P<0.05 was considered statistically significant. Results1.Observed under the fluorescence microscope, FAM-NC and inhibitor FAM-NC can transfect to Chang liver cells.2.The expression of Micro RNA-126 was significantly increased in Micro RNA-126 mimics transfection group compared with the normal control group(P<0.05).Compared with normal control group, there is no statistically significant difference of the relative expression of Micro RNA- 126 in the other groups(P>0.05).3.The expression of IRS-1、PIK3R2 were significantly decreased in Micro RNA-126 mimics transfection group compared with the normal control group(P<0.05). But there is no significant difference among the other groups(P>0.05).Compared with the normal control group, the phosphorylation level of Akt-2(p-Akt-2/Akt-2)was significantly decreased(P<0.05), there is no statistically significant difference of the phosphorylation level of Akt-2 in the other groups compared with the normal control group(P>0.05). ConclusionMicro RNA-126 affects glucose metabolism by inhibiting IRS-1/PIK3R2/ Akt-2 signal transduction pathway. |
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