Effects of miR-494 on the insulin signaling pathway.

<p>The mimic form of miR-494 was overexpressed in cells. Cells were starved for 4 h before treatment with 0, 10, 100 nM of insulin for 20 min, and the levels of p-Akt (Ser473 and Thr308), p-AS160, p-p70S6K, and p-GSK-3α/β were measured in C₂C₁₂ myoblasts (A) and CHO_IR/IRS-1 cells (D). Bars show densitometric quantitation of phosphorylations of Akt, GSK-3α/β, AS160 and p70S6K in C₂C₁₂ myoblasts (B) and CHO_IR/IRS-1 cells (E). (C) The level of p-IRS1 (Tyr608) was measured in insulin-treated C₂C₁₂ myoblasts. GAPDH and actin were used as loading controls. Levels of phospho-proteins between miR-494 transfected cells and control were compared at each concentration of insulin. The values are expressed as the means ± SEM. *P<0.05; **,P<0.01. (F) RT-PCR analysis of genes regulated by miR-494. Total RNA from cells transfected with miR-494-mimic or ASO was prepared from C₂C₁₂ cells and subjected to reverse transcription and PCR. The three lanes represent the same sample with triplicate loading. Fold induction indicates the ratio of expression levels of mRNA in mimic- or ASO-transfected cells compared to control. The glyceraldehyde-3-phosphate dehydrogenase gene (Gapdh) was used as a loading control.