Regulation of genes related to the insulin signaling pathway by miR-494.

<p>(A) The sequences of mature form of miR-494 were compared between species. The sequences of miR-494 were obtained from miRbase (release 20; <a href="http://mirbase.org" target="_blank">miRbase.org</a>). (B) Human HeLa cells were overexpressed with the mimic form of miR-494. Cells were starved for 4 h before treatment with 100 nM of insulin for 20 min. The levels of p-Akt (Ser473 and Thr308), p-AS160, p-p70S6K, and p-GSK-3α/β were measured (C). Bars show densitometric quantitation of phosphorylations of Akt, GSK-3α/β, AS160 and p70S6K. GAPDH and actin were used as loading controls. (C-E) PCR array analysis of genes related to insulin signaling pathway by miR-494 in HeLa cells. Real-time PCR was carried out using Human Insulin Signaling Pathway PCR, after transfection of miR-494-mimic oligonucleotide. (C) Intensity scatter plot comparing the mRNA profiles of miR-494 transfected cells and control cells. The 11 mRNAs that were up- or downregulated more than 1.3-fold are highlighted and labeled. (D) Genes that changed more than 1.5-fold or with significance (P<0.05) were clustered. Gene names are denoted on the left. Fold changes and p-values are denoted on the right side of the panel. (E) The percentages of significantly (P<0.05) up- or downregulated genes are compared to the control in each metabolic category. The number of genes are described inside the bar in each group. (F) RT-PCR analysis was performed in miR-494 mimic transfected HeLa cells for 9 genes; <i>Slc2A4, Slc2A1, Insr, Grb2, Gab1, Ptpn1, Jun</i> and <i>Ldlr</i> with <i>Gapdh</i> as the loading control. The three lanes indicate the same sample with triplicate loading. Fold induction indicates the ratio of expression levels of mRNA in mimic transfected cells compared to control. Gene abbreviations: <i>Slc2A4</i>, solute carrier family 2 member 4; <i>Slc2A1</i>, solute carrier family 2 member 1; <i>Insr</i>, insulin receptor; Grb2, growth factor receptor-bound protein 2; <i>Gab1</i>, Grb2-associated binding protein 1; <i>Ptpn1</i>, protein tyrosine phosphatase, non-receptor type 1; <i>Jun</i>, jun proto-oncogene and <i>Ldlr</i>, low density lipoprotein receptor. (G) Immunoblot analysis of Glut1 and Glut4 was performed in miR-494 mimic transfected HeLa cells. GAPDH was used as a loading control.