Figure 4

MiR-99a targeted IGF1R in OSCC cells. (A) Comparison of nucleotides in the miR-99a seed sequence and the targets, mTOR (upper panel) and IGF1R (lower panel), within the 3′UTR among species. (B) Significant negative correlation between the expression of miR-99a and IGF1R protein in OSCC cell lines (r = −0.747, p = 0.0207) by Pearson correlation. Average amounts of IGF1R protein were determined in two independent blots. *p < 0.05. (C) No significant correlation between expression of miR-99a and mTOR protein in OSCC cell lines by Pearson correlation (r = −0.1164, p = 0.7655). Average amounts of mTOR proteins were determined in two independent blots. (D) Immunoblot analysis of IGF1R and mTOR expression in OEC-M1 and SCC15 cells with ectopic miR-99a expression (OEC-M1 miR-99a and SCC15 miR-99a) or non-silencing microRNA expressing controls (OEC-M1 NS and SCC15 NS). The protein levels were normalized against an internal control α-tubulin. Ratios were determined by dividing the normalized protein levels in miR-99a expressing cells with that in non-silencing microRNA expressing controls. The means in the lower graph were measured by averaging the ratios from independent blots. Bar, SE; *p < 0.05. (E) Ectopic miR-99a expression suppressed the expression of the Renilla luciferase reporter gene containing the wild type miR-99a binding sequence in IGF1R 3′UTR (IGF1R 3′UTR) in OEC-M1 miR-99a cells. The luciferase activity of the construct with the deleted miR-99a binding site for IGF1R 3′UTR (ΔIGF1R 3′UTR) was assigned as 1. Bar, SE; *p < 0.05.