Figure 4.

Pim1 is a direct target of miR-101-3p. A. Real-time PCR analysis showed that Pim-1 expression is significantly elevated in ACC tissues (t-test, ***, P<0.001). GAPDH was used as the internal loading control. B. Correlation between miR-101-3p expression and Pim-1 expression (P<0.038, r²=0.1132). C. Predicted miR-101-3p target sequences in 3’UTR of Pim-1 and its mutant (MUT) containing 4 mutated nucleotides. The wild type (WT) or mutant (MUT) reporter plasmid was co-transfected with miR-101-3p mimics or the negative control (NC) into SACC-LM or SACC-83 cells. Renilla luciferase vector was used as the internal control (t-test, **, P<0.01. ***, P<0.001, ns=no significance). D, E. Protein levels of Pim-1, survivin, cyclin D1, and β-catenin in SACC-83 and SACC-LM cells transfected with miR-101-3p mimics or miR-101-3p inhibitor (anti-miR-101-3p) were determined by using Western blot analysis. Quantification was carried out by using Image J through pixel analysis of bands with normalization to GAPDH as a loading control. Error bars indicate mean ± S.D.