Fig. 2: Identification of pathway(s) commonly targeted by downregulated miRNAs and validation of targets. | Cell Death Discovery

Fig. 2: Identification of pathway(s) commonly targeted by downregulated miRNAs and validation of targets.

From: Sponging of five tumour suppressor miRNAs by lncRNA-KCNQ1OT1 activates BMPR1A/BMPR1B-ACVR2A/ACVR2B signalling and promotes chemoresistance in hepatocellular carcinoma

Fig. 2

(A) Graphical representation of KEGG pathway analysis with validated target genes of five miRNAs. (B) Integrated analysis of the target genes of the five miRNAs using miRNet. Expression analysis of target genes by qRT-PCR using (C) liver tissues of HCV and HBV infected HCC patients vs. control, and (D) Huh7 cells transfected with HCV (pS52/JFH1) and HBV (pSV2neoHBV2x) producing plasmids for 48 h. (E) The expression of target genes were determined in Huh7 cells transfected with pRNAU6.1 (vector), pScramble Pre-miRNA (Scr. miRNA), pPre-miRNA (miRNA), pPre-miRNA + scramble anti-miRNA oligo (Scr. anti-miRNA), or pPre-miRNA + anti-miRNA oligo (anti-miRNA) independently. (F) 3′UTR-Luciferase assay using Huh7 cells transfected with either wild-type-3′UTR-Luciferase construct (3’UTR-wt) alone or with pScramble pre-miRNA, pPre-miRNA, pPre-miRNA + scramble anti-miRNA oligo, pPre-miRNA + anti-miRNA oligo, separately and mutated-3′UTR-luciferase construct (3′UTR-mut) + pPre-miRNA. (G) Immunoblot analysis with anti-ACVR2A, anti-BMPR1B, anti-BMPR1A, and anti-GAPDH-HRP using lysates of Huh7 cells transfected with pRNAU6.1, pPre-miRNA, pPre-miRNA + anti-miRNA oligo, and pPre-miRNA + scramble anti-miRNA oligo. p value was calculated using Mann–Whitney test for C and unpaired student’s t-test for D–F. *, **, *** indicate p-value < 0.05, <0.01, and <0.001 respectively. ns means not significant.

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