Hepatitis B virus X protein promotes hepatoma cell proliferation via upregulation of MEKK2

Abstract

Aim: To investigate the mechanism underlying the increase of hepatoma cell proliferation by hepatitis B virus X protein (HBx).

Methods: HepG2, H7402 and HepG2.2.15 cells, which constitutively replicated hepatitis B virus were used. The effects of HBx on hepatoma cell proliferation were examined using 5-ethynyl-2-deoxyuridine (EdU) incorporation assay and MTT assay. The expression level of MEKK2 was measured using RT-PCR, Western blot and luciferase reporter gene assay. The activity of activator protein 1 (AP-1) was detected using luciferase reporter gene assay. The phosphorylation levels of JNK and c-Jun were measured using Western blot. The expression levels of HBx and MEKK2 in 11 clinical hepatocellular carcinoma (HCC) tissues were measured using real time PCR and Western blot. In addition, the expression of MEKK2 in 95 clinical HCC tissues was examined using immunohistochemistry.

Results: HBx significantly enhanced HepG2-X cell proliferation. In HepG2-X, H7402-X and HepG2.2.15 cells, the expression level of MEKK2 was remarkably increased. In HepG2.2.15 cells, HBx was found to activate JNK and AP-1, which were the downstream effectors of MEKK2 in HepG2-X and HepG2.2.15 cells. In 11 clinical HCC tissues, both HBx and MEKK2 expression levels were remarkably increased, as compared to those in the corresponding peritumor tissues. In 95 clinical HCC tissues, the rate of detection of MEKK2 was 85.3%.

Conclusion: HBx promotes hepatoma cell proliferation via upregulating MEKK2, which may be involved in hepatocarcinogenesis.

Figure 1

HBx enhances the proliferation of HepG2-X cells. (A, B) The proliferation ability of hepatoma cells was tested using the 5-ethynyl-2-deoxyuridine (EdU) incorporation and MTT assays. Results are representative of three independent experiments. Values represent mean±SD. ^bP<0.05, ^cP<0.01, student's t test.

Figure 2

HBx upregulates the expression of MEKK2. (A) The levels of MEKK2 and HBx were detected using RT-PCR and immunoblot analysis. GAPDH and β-actin were used as internal controls. (B) The levels of MEKK2 and HBx were detected in HepG2.2.15 cells using immunoblot analysis. (C) The plasmids of pGL3-MEKK2 pro and pGL3 basic vector were transfected into HepG2 and H7402 cells, respectively. The relative luciferase activity was tested using the luciferase reporter gene assay. (D) The promoter activities of MEKK2 were examined in HepG2-X/H7402-X cells. (E) The promoter activities of MEKK2 were measured after treatment with RNAi that targeted HBx mRNA in HepG2-X/H7402-X/HepG2.2.15 cells in a dose-dependent manner. Results are representative of three independent experiments. Values represent mean±SD. ^bP<0.05, ^cP<0.01, student's t test.

Figure 3

HBx upregulates the expression of MEKK2 to mediate AP-1 and JNK activation. (A) The activity of AP-1 was determined in HepG2.2.15 cells using the luciferase reporter gene assay after HBx RNAi treatment. (B) The phosphorylation levels of JNK and c-Jun were determined using immunoblot analysis. (C) The activity of AP-1 was determined in HepG2.2.15 cells using the luciferase reporter gene assay after MEKK2 RNAi treatment. (D) The phosphorylation levels of JNK and c-Jun were determined using immunoblot analysis. The results are representative of three independent experiments. Values represent mean±SD. ^cP<0.01, student's t test.

Figure 4

MEKK2 contributes to HBx-mediated growth of hepatoma cells. (A, B) The proliferation ability of HepG2-X cells was tested using the EdU incorporation and MTT assays after MEKK2 siRNA treatment. (C, D) The proliferation ability of HepG2.2.15 cells was tested using the EdU incorporation and MTT assays after MEKK2 siRNA treatment. Results are representative of three independent experiments. Values represent mean±SD. ^bP<0.05, ^cP<0.01 vs NC, student's t test.

Figure 5

MEKK2 is overexpressed in clinical HCC tissues. (A, B) The relative mRNA expression levels of HBx and MEKK2 were examined in clinical HBx-positive HCC tissues using real time PCR. Values represent mean±SD. ^bP<0.05, ^cP<0.01, Student's t test. (C) The expression levels of HBx and MEKK2 were examined in HCC patients using Western blot analysis. Protein bands were quantified using Quantity One software (Bio-Rad). The value under each pair of samples (T/P), represents the ratio of HBx or MEKK2 expression in the HCC tissue to that in its corresponding peritumor tissue and indicates the fold change in the protein level in HCC. P, peritumor tissue; T, HCC tissue. (D) Immunohistochemical staining showed the expression levels of MEKK2 in HCC tissues using a tissue microarray. (a) Negative control; (b) MEKK2-positive staining. Imaged at 200× magnification.