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Fig. 4 | Cellular & Molecular Biology Letters

Fig. 4

From: Hsa-microRNA-27b-3p inhibits hepatocellular carcinoma progression by inactivating transforming growth factor-activated kinase-binding protein 3/nuclear factor kappa B signalling

Fig. 4

TAB3 is the downstream target of hsa-miR-27b. A1, A2 qRT-PCR (A1) and western blot (A2) analysis of TAB3 mRNA and protein levels after overexpression or knockdown of hsa-miR-27b in HCC cells. Data were normalized with GAPDH and are shown as the fold change relative to the control cells. GAPDH was used as a loading control for western blot analysis (n = 3 for qRT-PCR, Mann–Whitney test). B MHCC97H cells were transfected with hsa-miR-27b mimics at the indicated concentrations. qRT-PCR analysis of TAB3 mRNA (top) and western blot analysis of TAB3 protein levels (bottom) were performed. Data were normalized to GAPDH and are shown as fold change compared with the cells treated with an NC for qRT-PCR (n = 3, one-way ANOVA). GAPDH was used as a loading control for western blot analysis. C Relative TAB3 expression in HCC tissues (n = 43) compared with ANTs (n = 43) analysed by western blots. D Linear regression analysis between relative TAB3 mRNA (left) or protein levels (right) and hsa-miR-27b expression in HCC specimens (n = 43). E RNA immunoprecipitation assays of 3′UTR-TAB3 enrichments by anti-IgG or anti-AGO2 antibodies in the hsa-miR-27b-overexpressing MHCC97H cells (top) or the hsa-miR-27b-knockdown Hep3B cells (bottom). Data are shown as percentage of 10% input (n = 3, Kruskal–Wallis test). F Schematic diagram of hsa-miR-27b binding sequences in the 3′UTR of TAB3 and mutants in the 3′UTR of TAB3 (TAB3-MUT) (top). Dual luciferase reporter assay of MHCC97H and Hep3B cells co-transfected with wild-type (WT) or mutated TAB3 3′UTR luciferase reporter plasmids with hsa-miR-27b mimics or inhibitors (n = 3, Mann–Whitney test). A, B, E and F were repeated three times. *p < 0.05. **p < 0.01. Data are shown as mean ± SD. NC, negative control; ANT: adjacent non-tumourous tissues; HCC, hepatocellular carcinoma; 3′UTR, 3′ untranslated region; ctrl, control; KD, knockdown

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