Figure 3

miR-218-5p directly targets the 3′-UTR of PRKCE and downregulated its expression. (a) PRKCE and SFMBT1 were potential targets of miR-218-5p in all six miRNA target prediction algorithms. (b, c) Q-PCR to detect PRKCE and SFMBT1 mRNA level in GBC cells when transfected with miR-218-5p mimic or antagomir. n=3; bar, S.E.M. (d) Western blot to analyze PRKCE and SFMBT1 protein levels in GBC cell lines when transfected with miR-218-5p mimic or antagomir. (e) A schematic diagram showing the predicted miR-218-5p binding sites and the designed mutant sequence in the 3′-UTR of PRKCE (up), and the luciferase reporter constructs (down). (f, g) Firefly luciferase activity analysis of PRKCE 3′-UTR performed after co-transfection with PRKCE-wild type or PRKCE-mutant pGL3 constructs and miR-218-5p mimic GBC cell lines. n=3; bar, S.E.M. (h) Q-PCR analysis of PRKCE mRNA levels in 36 pairs of GBC and CNG tissues. (i) Semi-quantitative analysis and the representative images (× 400) of IHC staining for PRKCE protein in 82 paired GBC and CNG FFPE tissues. (j) The correlation between miR-218-5p and PRKCE expression in 36 GBC tissues measured by Q-PCR. (k) POS analysis based on PRKCE protein expression levels in 82 GBC patients. GAPDH was used to normalize the Q-PCR results, and β-actin was the loading control in western blot assay. NS, not significant, *P<0.05; **P<0.01; ***P<0.001; Student’s t-test