HBV infection exacerbates PTEN defects in hepatocellular carcinoma through upregulation of miR-181a/382/362/19a

Abstract

A high hepatitis B virus (HBV) load and chronic hepatitis B infection are well-recognized risk factors for the development of hepatocellular carcinoma (HCC), highlighting the need for research into the mechanisms underlying the role of HBV infection in HCC. Because phosphatase and tensin homolog (PTEN) has been implicated in HCC development, we explored whether PTEN has a role in HBV-related hepatocarcinogenesis. We found that PTEN expression was correlated with advanced clinicopathological features and that HBV infection exacerbates PTEN defects in HCC. Using an integrated approach, we then investigated if miRNAs linked HBV infection to PTEN downregulation in HCC and found that PTEN was a target of miR-181a/382/362/19a. We also show that miR-181a/382/362/19a-mediated inhibition of PTEN led to an enhanced malignant phenotype and stimulation of AKT signaling in HCC cells. Collectively, our results indicate that HBV infection exacerbates PTEN defects in hepatocellular carcinoma through upregulation of miR-181a/362/382/19a. Our work implicates miR-181a/362/382/19a and PTEN as potential biomarkers and targets for novel prognostic, diagnostic, and therapeutic strategies targeting HBV-related HCC.

Keywords: HBV; hepatocellular carcinoma; microRNA; phosphatase and tensin homolog deleted on chromosome 10.

Figure 1

HBV infection decreased PTEN expression in HCC tissues and cell lines. A. PTEN expression in cancerous (Ca) and paracancerous (CaP) tissues from HCC patients was detected by immunohistochemistry (IHC). Hepatic hemangioma tissues were used as controls. Images were obtained using a microscope at × 400 magnification. Three random fields of view were used to calculate mean densities of PTEN-positive areas with Image-Pro Plus software. B. PTEN expression in poorly differentiated (PD), moderately differentiated (MD), and well differentiated (WD) cancerous tissues and hepatic hemangioma tissues (control) detected by IHC. C. PTEN expression in Ca and CaP tissues with HBV infection (HBV+) or without HBV infection (HBV-) detected by IHC. D. PTEN expression in HBsAg+ and HBsAg- cancerous tissues detected by IHC. E. PTEN expression in the cancerous tissues with a high HBV cccDNA load (> 1 × 10⁴) or low HBV cccDNA load (< 1 × 10⁴) detected by IHC. F. PTEN expression in LO2, HepG2, and HepG2.215 cells was detected by quantitative real-time PCR (left) and western blot (middle and right). The mRNA expression was normalized to that of GAPDH and protein expression was normalized to that of β-actin. PTEN expression values in LO2 cells were designated as 1 and data were expressed as fold changes. Data represent means ± standard deviation from triplicate experiments. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 2

HBV infection increased miR-181a/362/382/19a levels in HCC tissues and cell lines. A. The expression levels of miR-181a/362/382/19a in LO2, HepG2, and HepG2.215 cells were determined by quantitative real-time PCR and normalized to that of U6. Data represent means ± standard deviation from triplicate experiments. *P < 0.05; **P < 0.01; ***P < 0.001. B. The expression levels of miR-181a/362/382/19a in cancerous tissues with HBV infection (HBV+) or without HBV infection (HBV-) were determined by quantitative real-time PCR and normalized to that of U6. The median of relative miR-181a/362/382/19a values was included for subgroup separation for both HBV+ and HBV- cancerous tissues. Differences in miR-181a/362/382/19a expression levels were evaluated by the chi-squared test. (miR-181a: χ² = 16.68, df = 1, P < 0.0001; miR-362: χ² = 29.89, df = 1, P < 0.0001; miR-382: χ² = 25.93, df = 1, P < 0.0001; miR-19a: χ² = 42.37, df = 1, P < 0.0001).

Figure 3

PTEN is a target of miR-181a/362/382/19a. Diagrams show the putative binding sites (red) for miR-181a (A), miR-362 (B), miR-382 (C) and miR-19a (D) in PTEN. The wild-type (WT) 3’UTR of PTEN is shown in blue and the corresponding mutant (MUT) sites in green. Bar graphs show the relative fluorescence intensity of HepG2 cells transfected with dual-luciferase reporter vectors containing either the WT or MUT 3’UTR of PTEN plus miR-181a/362/382/19a mimics (miR-181a/362/382/19a-m) or negative control mimics (NC-m). Data represent means ± standard deviation from triplicate experiments. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 4

MiR-181a/362/382/19a downregulated PTEN expression in HepG2 cells. The mRNA (left) and protein (middle and right) expression of PTEN in HepG2 cells transfected with miR-181a (A), miR-362 (B), miR-382 (C), miR-19a (D) mimics or negative control mimics (NC-m) were examined by quantitative real-time PCR and western blot. The mRNA expression levels were normalized to that of GAPDH and protein expression levels were normalized to that of β-actin. Values of PTEN expression were designated as 1 and data were expressed as fold changes. Data represent means ± standard deviation from triplicate experiments. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 5

MiR-181a/362/382/19a enhanced the activities of HCC cells, and overexpression/silencing of PTEN antagonized/mimicked their effects. HepG2 cells were transfected with miR-181a/362/382/19a mimics (miR-181a/362/382/19a-m) or negative control mimics (NC-m) plus PTEN, shPTEN or control vectors. HepG2.215 cells were transfected with miR-181a/362/382/19a inhibitors (miR-181a/362/382/19a-i) or negative control inhibitors (NC-i) plus PTEN, shPTEN or control vectors. A. Proliferation of HepG2 (top) and HepG2.215 (bottom) cells was determined by CCK-8 assay. B. Migration of HepG2 (top) and HepG2.215 (bottom) cells was assessed by Transwell assay. Representative images and bar graphs are shown. C. Migration of HepG2 (left) and HepG2.215 (right) cells was examined by wound-healing assay. Representative images at the indicated time points following scratch initiation and bar graphs are shown. Values at the 0 h-timepoint were designated as 1 and the rate of closure was assessed. D. Invasion of HepG2 (top) and HepG2.215 (bottom) cells was determined by Transwell-Matrigel assay. Representative images and bar graphs are shown. Data represent means ± standard deviation from triplicate experiments. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 6

MiR-181a/362/382/19a upregulated AKT phosphorylation, and overexpression/silencing of PTEN antagonized/mimicked their effects. A-D. HepG2 cells were transfected with miR-181a/362/382/19a mimics (miR-181a/362/382/19a-m) or negative control mimics (NC-m) plus PTEN, shPTEN or control vectors. HepG2.215 cells were transfected with miR-181a/362/382/19a inhibitors (miR-181a/362/382/19a-i) or negative control inhibitors (NC-i) plus PTEN, shPTEN or control vectors. The expression levels of PTEN, phosphorylated AKT (p-AKT), and total AKT were determined by western blot.