Downregulation of HBx Restrains Proliferation, Migration, and Invasion of HepG2 Cells

Abstract

Liver cancer is a major contributor to cancer-related death with poor survival for sufferers. Meanwhile, Hepatic B virus X protein (HBx) and XB130 are likely to participate in the pathogenesis of liver cancer. However, the detailed mechanism of HBx/XB130 in liver cancer remains to be further investigated. Our study explored the effects of HBx/XB130 on liver cancer progression. HBx and XB130 expression was detected by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blot. Overexpression of HBx and XB130 was found in liver cancer tissues and cells. Mechanistic study revealed that HBx could bind to and positively regulate XB130 in HepG2 cells. Subsequently, HBx expression was knocked down, while XB130 was overexpressed in HepG2 cells in order to observe the specific role of HBx/XB130 in liver cancer in vitro. Results of CCK-8, Transwell, wound healing, and colony formation assays suggested that HBx could mediate biological function of HepG2 cells by activating the XB130-mediated PI3K/AKT pathway. In summary, our data illustrate that inhibition of HBx effectively suppressed proliferation and metastasis and induced apoptosis of liver cancer cells, which might be partially reversed by XB130. HBx and XB130 may be potential targets for liver cancer pathogenesis.

Figure 1

Expression of HBx and XB130 in liver cancer tissues and the matched adjacent normal tissues. (a) Differentially expressed mRNAs in liver cancer tissues and adjacent normal tissues screened out using microarray-based analyses. (b, c) Expression of HBx and XB130 in liver cancer and adjacent tissues examined by RT-qPCR. (d, e) Correlation analyses on HBx and XB130 expression with tumor size. (f) HBx and XB130 expression in tissues analyzed by immunohistochemistry. (g, h) Survival analysis on HBx and XB130 expression with prognosis of liver cancer patients. ^∗p < 0.05 indicates statistical significance.

Figure 2

Expression of HBx/XB130 in liver cancer cells and normal hepatocytes. (a) Expression of HBx/XB130 in HepG2 and MIHA cells determined by RT-qPCR. (b) Protein levels of HBx/XB130 in HepG2 and MIHA cells analyzed by Western blot analysis. (c, d) Transfection efficiency of pcDNA 3.1 plasmids containing si-HBx or XB130-OE evaluated by RT-qPCR. ^∗p < 0.05 and ^∗∗p < 0.01.

Figure 3

Binding relation of HBx with XB130 in HepG2 cells. (a, b) mRNA and protein expression of HBx and XB130 in HepG2 cells with high expression of XB130 and low expression of HBx determined by RT-qPCR and Western blot analysis. (c) Correlation of XB130 with HBx in liver cancer tissues. (d) Binding relation between HBx and XB130 analyzed by RIP. ^∗#p < 0.05.

Figure 4

HBx/XB130 axis facilitates proliferation of HepG2 cells. (a) Transfection efficiency of cells with silenced HBx and overexpressed XB130 detected by RT-qPCR. (b) Proliferative ability of cells examined by CCK-8 assay. (c) Colony formation ability of cells examined by colony formation assay. ^∗#p < 0.05.

Figure 5

HBx promotes migration/invasion of HepG2 cells by upregulating XB130. (a) Cellular migration assessed by wound healing assays. (b) Number of invasive cells determined by Transwell assay. ^∗#p < 0.05.

Figure 6

HBx suppresses apoptosis of HepG2 cells by increasing XB130 expression. (a) Cell apoptosis examined using flow cytometry. (b) Cell cycle determined using flow cytometry. ^∗#p < 0.05.

Figure 7

HBx activates the PI3K/AKT pathway by upregulating XB130, which is determined by Western blot analysis. ^∗#&p < 0.05.

Figure 8

Schematized analysis showing that HBx promotes activity of PI3K/AKT pathway by binding to XB130, which enhanced proliferation, migration, and invasion of HepG2 cells and curtailed cellular apoptosis.