MiR-148b suppresses cell proliferation and invasion in hepatocellular carcinoma by targeting WNT1/β-catenin pathway

Abstract

Accumulating evidences indicate that microRNAs play a vital role in regulating tumor progression. However, the roles of miR-148b in hepatocellular carcinoma (HCC) are still largely unknown. In this study, our data showed that miR-148b was significantly downregulated in 40 pairs of human HCC tissues. Further, the deregulated miR-148b was significantly correlated with larger tumor size, more tumor number, metastasis and worse prognosis in HCC. Overexpression of miR-148b inhibited HCC HepG2 cells proliferation and tumorigenicity. Further, miR-148b induced cells apoptosis by activating caspase- 3 and caspase-9, and induced S phase arrest by regulating cyclinD1 and p21, and also inhibited cell invasion. Data from the dual-luciferase reporter gene assay showed that WNT1 was a direct target of miR-148b, and overexpressed WNT1 inversely correlated with miR-148b levels in HCC tissues. Silencing of WNT1 inhibited the growth of HCC cells, and also induced cells apoptosis and inhibited invasion, which is consistent with the effects of miR-148b overexpression. MiR-148b downregulated expression of WNT1, β-catenin and C-myc, while upregulated E-cadherin expression. We conclude that the frequently downregulated miR-148b can regulate WNT1/β-catenin signalling pathway and function as a tumor suppressor in HCC. These findings suggest that miR-148b may serve as a novel therapeutic target for HCC.

Figure 1. Analysis of miR-148b expression in human HCC tissues and survivals of HCC patients.

(A) The relative expression levels of miR-148b in human HCC tissues (n = 40) and matched normal liver tissues (n = 40) were detected by qRT-PCR. U6 was used as the control for RNA loading, and miR-148b abundance was normalized to U6 RNA. (B) Kaplan- Meier curves of overall survivals of 40 HCC patients, according to miR-148b expression scored as low expression (below the median value, n = 20) and high expression level (above the median value, n = 20). MiR-148b downregulation correlated significantly with shorter overall survivals. P-value was shown with log-rank test. **P<0.01.

Figure 2. MiR-148b inhibited proliferation of HCC cells in vitro and tumorigenicity in vivo.

(A) The effect of transient transfection of miR-148b mimics (miR-148b) or miR-138 inhibitor (anti-miR-148b) and their respective negative control (miR-NC or anti-miR-NC) (50 nM) for 48 h on the growth of HepG2 cells was examined by MTT assay. (B) The effect of miR-148b on proliferation of HepG2 cell was measured by colony formation assay. The colonies were evaluated according to the ratio between lentiviral miR-148b (LV-miR-148b)-infected cells and empty lentiviral vector (LV-NC)–infected cells at a multiplicity of infection of 10. Each data is representative of three independent experiments. The LV-miR-148b or LV-NC transfected HepG2 cells were injected subcutaneously into nude mice, respectively. Excised tumor volumes (C) at 36 days after implantation and tumor growth curves (D) were shown as indicated. Data were shown as the Mean ± SD of 10 mice. *P<0.05; **P<0.01.

Figure 3. The effects of miR-148b on in vitro apoptosis, cell cycle arrest and invasion of HCC cells.

After transfection with miR-148b or miR-NC (50 nM) for 48 h, (A) the apoptosis of HepG2 cells was measured by Annexin V staining and flow cytometry (the right lower quadrant represents apoptosis), and (B) representative western blotting images for detecting the protein levels of Pro-caspase-3 and Pro-caspase-9, cleaved caspase-3 and caspase-9. GAPDH served as the loading control. HepG2 cells were treated as above, cell cycle distribution was measured by PI staining and flow cytometry (C) and cell cycle protein cyclinD1 and p21expression (D). (E) HepG2 cells were treated as above, and the cell invasion ability was measured by matrigel invasion assays. Data were representative of three independent experiments. *P<0.05; **P<0.01.

Figure 4. WNT1 was the direct target of miR-148b in HCC cells.

(A. Top) Human WNT1 3′UTR fragment containing wild-type or mutated miR-148b–binding sequence. (WT: wild type; MUT: mutant type). (A. Bottom) MiR-148b and its putative binding site in the 3′UTR of WNT1. (B) Luciferase reporter assays, with co-transfection of wild-type or mutant 3′UTR (100 ng) and miR-148b or miR-NC (50 nM) in HepG2 cells as indicated. Firefly luciferase activity was normalized by Renilla luciferase activity. After transfection with miR-148b or anti-miR-148b (50 nM) for 48 h, qRT-PCR (C) and western blot (D) were used for monitoring WNT1 expression in HepG2 cells. *P<0.05; **P<0.01.

Figure 5. WNT1 inversely correlated with miR-148b levels in HCC tissues.

(A) WNT1 was detected in 40 HCC tissues and matched normal liver tissues by qRT-PCR, and the WNT1 abundance was normalized to GAPDH. (B) The relationship between WNT1 and miR-148b expression was explored by Spearman's correlation analysis in HCC tissues. *P<0.05; **P<0.01.

Figure 6. MiR-148b targeted WNT1 contributed to HCC cells growth.

(A) The WNT1expression was examined by western blotting in HepG2 cells transfected with 3 different siRNAs targeting WNT1 (si-WNT1-1, si-WNT1-2 and si-WNT1-3) or NC. (B) The effect of transfection of si-WNT1 and anti-miR-148b targeting WNT1 in HepG2 cells was examined by western blotting at 48 h following transfection. (C) The effect of anti-miR-148b on si-WNT1-induced proliferation inhibition of HepG2 cells was examined by MTT assays 48 h following transfection. All results were reproducible in three independent experiments. *P<0.05, **P<0.01.

Figure 7. The effects of miR-148b on the downstream signaling pathway of WNT1.

HepG2 cells were transfected with miR-148b or miR-NC for 48 h, the expression levels of β-catenin, C-myc and E-cadherin proteins in HepG2 cells were detected by western blotting. The results were standardized against the levels of GAPDH and repeated three times, *P < 0.05, **P < 0.01.