RESUMO
Objective To investigate the expression of miRNA-802 (miR-802) and the relationship with glucose metabolism and insulin sensitivity in insulin resistant skeletal muscle cells. Methods L6 rat myoblasts were recovered, cultured and differentiated into L6 muscle cells, incubated with 0.4 mmol/L palmitic acid (PA) and then randomly divided into two groups: Control group (Con) and PA group (PA). After 24 hours, the glucose concentration in the culture medium was measured to determine whether the model was constructed successfully. After successful establishment of the insulin resistance L6 rat myoblasts model, then the cells were transfected with miR-802 mimic or inhibitor to regulate the expression of miR-802. The experimental groups were divided into 5 subgroups as follows: control group, PA group, PA group infected with miR-802 mimic (PA+miR-802-mimic), PA group infected with miR-802 inhibitor (PA+miR-802-inhibitor), and PA group infected with negative control (PA+negative control). The mRNA and protein expressions of the insulin signaling pathway [including phosphatidylinositol-3kinase (PI3K), protein kinase B (Akt), and glucose transporter 4 (GluT4)] were examined by RT-PCR and Western blotting. Results Compared with control group, the mRNA expression in miR-802 group increased significantly (1.458±0.264 vs. 3.108±0.513). Compared with PA group and PA+negative control group, the mRNA expression in miR-802 group increased obviously in PA+miR-802 mimic group (3.108±0.513, 3.442±0.104 vs. 11.743±0.933), and the mRNA expressions of PI3K (0.724±0.032, 0.682±0.059 vs. 0.494±0.025), Akt (0.819±0.044, 0.718±0.033 vs. 0.754±0.028) and GluT4 (0.719±0.038, 0.666±0.056 vs. 0.427±0.031) significantly decreased, and the protein expression of p-PI3K/PI3K (0.349±0.056, 0.351±0.019 vs. 0.195±0.026), p-Akt/Akt (0.639±0.002, 0.557±0.04 vs. 0.261±0.075)and GluT4 (0.648±0.028, 0.590±0.026 vs. 0.413±0.096) significantly decreased (P<0.01). Compared with PA and PA+negative control group, the mRNA expression in miR-802 group decreased in PA+miR-802 inhibitor group (3.108±0.513, 3.442±0.104 vs. 1.069±0.056), the mRNA expressions of PI3K (0.724±0.032, 0.682±0.059 vs. 0.887±0.016), Akt (0.819±0.044, 0.718±0.033 vs. 0.814±0.026) and GluT4 (0.719±0.038, 0.666±0.056 vs. 0.908±0.054) significantly increased, and the protein expressions of p-PI3K/PI3K (0.349±0.056, 0.351±0.019 vs. 0.494±0.012), p-Akt/Akt (0.639±0.002, 0.557±0.04 vs. 0.951±0.031) and GluT4 (0.648±0.028, 0.590±0.026 vs. 0.756±0.007) significantly increased (P<0.01). Conclusion miR-802 regulates insulin sensitivity and glucose metabolism in L6 muscle cells, so inhibition of miR-802 expression may be a potential therapeutic target for type 2 diabetes.
RESUMO
@#To investigate the effect of miR-802 on insulin secretion by islet β cells and its mechanism, miR-802 was overexpressed or knocked down in primary islet cells and Min6 cells via transfecting miR-802 mimic and miR-802 inhibitor, respectively. The effect of miR-802 on insulin secretion was detected by ELISA. The target gene of miR-802 was confirmed by miRNA target gene database prediction, luciferase report and Western blot. The function recovery experiment was carried out to clarify the mechanism of miR-802 regulating β cell secretion of insulin. The results showed that overexpression of miR-802 in islet primary cells and Min6 cells inhibited insulin secretion. qPCR and Western blot showed that miR-802 inhibited insulin secretion by inhibiting the transcription and translation of the target gene, hepatocyte nuclear factor 1β(Hnf1B).