Figure 4: The expression of miR-18a in CRC, and the regulation of miR-18a on the PIAS3 gene and STAT3 signaling pathway.

(A) Relative miR-18a expression levels were examined by qRT-PCR in 68 CRC tissues and adjacent normal tissues. *P < 0.05, paired t-test. (B) The linear correlations between the expression levels of CASC2 and miR-18a in CRC tissues (R² = 0.334, P < 0.001). The data were obtained using the logistic regression analysis. The luciferase reporter plasmids (RLuc-PIAS3-WT and RLuc-PIAS3-MT) containing the wild type 3′UTR region or mutant 3′UTR region of PIAS3 were co-transfected into CACO2 cells (C) and HT-29 cells (D) with miR-18a mimics or in parallel with the luciferase reporter vector psiCHECK2-CASC2-WT. Error bars are representative of Mean ± SD (n = 3). *P < 0.05 was calculated using Two-side Student’s t-test. (E) miR-18a was transfected into CRC cell lines, and the mRNA and protein levels of PIAS3 were evaluated by qRT-PCR (left) and western blot (right) 48 hours after transfection. β-actin was used as a loading control. (F) Luciferase reporter assay was performed to examine the effect of miR-18a on STAT3 transcriptional activity in CRC cells treated with miR-18a or negative control. Data are mean ± SD (n = 3). *P < 0.05, Two-side Student’s t-test. QRT-PCR analysis of STAT3 (G), Survivin (H) and c-Myc (I) expression in CACO2 and HT-29 cells transfected with miR-18a mimics or negative control. The data are presented as the mean ± SD (n = 3), *P < 0.05; Two-sided Student’s t-test. (J) Western blot analysis was conducted to evaluate the effect of miR-18a on the regulation of the STAT3 signaling pathway, including STAT3, pSTAT3, Survivin and c-Myc.