Regulation of putative target genes by miR-494.

<p>(A) The secondary structure of mouse premature(pre)-miR-494 (miRbase, Accession MI0003532). (B) Alignment of the seed matches in conserved sites of <i>Stxbp5, Igf1r, Rab35, Irs1</i> genes at 3’ untranslated region (3’ UTR) and seed types of each target gene. Entrez accession numbers and binding position at 3’ UTR are addressed for the representative transcript of each gene. Complementary sequences binding to miR-494 are shown in bold letters, with the m8 and A1 extensions highlighted in bold italic letters. Gene abbreviations: <i>Stxbp5</i>, syntaxin binding protein 5; <i>Igf1r</i>, insulin-like growth factor 1 receptor; Irs1, insulin receptor substrate 1 (C) RT-PCR analysis for predicted target genes in miR-494 mimic- or miR-494 ASO-transfected C₂C₁₂ cells. <i>Gapdh</i> gene was used as a loading control. (E) Immunoblot analysis of molecules related to the insulin signaling pathway. Cells were transfected with miR-494 mimic and subjected to immunoblot analysis. Antibodies for PTEN, p-PTP1B (S50) and IRS-1 were used. GAPDH was used as a loading control. The values were expressed as the means ± SEM and compared between miR-494 mimic transfected cells and control. *P<0.01.