Hepatitis B Virus X Protein Upregulates SREBP2 to Modulate Autophagy in Hepatocellular Carcinoma

Abstract

Background: The interaction between Hepatitis B virus X protein (HBx) and sterol regulatory element binding protein 2 (SREBP2) in modulating autophagy to influence inflammation and tumorigenesis is not fully understood. This research seeks to clarify the regulatory role of HBx in hepatocyte autophagy through SREBP2.

Methods: The study employed TCGA and GEO databases to investigate the expression of SREBF2 and autophagy-related proteins in liver cancer. Various experimental techniques, including dual-luciferase reporter assays, immunohistochemistry, Western blotting, immunofluorescence, GFP-mRFP-LC3 puncta analysis, transmission electron microscopy, and Fillipin III staining, were conducted on HBV-associated liver cancer tissues, HBV transgenic mice, and several liver cancer cell lines to assess the levels of HBx, SREBP2, autophagy, and cholesterol, respectively, as well as to explore potential associations between these factors.

Results: Bioinformatics analysis suggested up-regulation of SREBP2 and autophagy-associated genes in HBV-associated liver cancer. Elevated levels of cholesterol, SREBP2, and autophagy flux were detected in HBV-associated liver cancer tissues as compared to adjacent tissues. HBV transgenic mice had higher cholesterol, SREBP2, and autophagy levels than wild-type mice. HBx activated the SREBP2 promoter to enhance its transcription and nuclear translocation. HBx knockdown down-regulated SREBP2 expression and nuclear translocation levels in HepG2.2.15-siHBx cells. HepG2.2.15 and HepG2-HBx showed more autolysosomes than HepG2 cells; furthermore, HepG2.2.15-siHBx cells had fewer autolysosomes than HepG2.2.15 cells.

Conclusions: This research highlights that HBx upregulates SREBP2 and increases autophagic flux, accompanied by changes in cholesterol metabolism, which offers an additional theoretical foundation to elucidate that chronic HBV infection causes abnormal lipid metabolism and induces tumorigenesis.

Keywords: Hepatitis B virus X protein; autophagy; cholesterol; hepatocellular carcinoma; sterol regulatory element binding protein 2.

FIGURE 1

The expression of SREBF2 in HBV‐associated liver cancer based on TCGA and GEO databases. (A) Expression of cholesterol metabolism‐related genes and autophagy‐related genes at transcriptional levels in the TCGA database. (B) Using TCGA pan‐cancer datasets, SREBF2 expression was further compared between hepatitis B‐related liver cancer tissues and non‐hepatitis B‐related liver cancer tissues. (C) According to GEO datasets GSE83148 and GSE121248, SREBF2 expression was compared between cancerous and normal tissues as well as between hepatitis B‐related hepatitis tissues and normal liver tissues. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 2

Up‐regulation of SREBP2 and cholesterol levels and increased autophagy in HBV‐associated liver cancer tissues. (A) Immunohistochemical detection of LC3B, P62, Beclin1, SREBP2 and HBx expression in liver cancer and corresponding adjacent tissues of patients, with microscopic fields of view (200×); the average cumulative positive rate (IOD/area) of images were calculated by Image J software and analyzed by paired t‐tests; the values were expressed in mean ± SD. (B) Detection of SREBP2 expression and autophagy‐associated proteins in liver cancer and adjacent pathological tissues of four patients with concurrent HBV infection. (C) Detection of cholesterol levels in liver cancer and adjacent pathological tissues of patients with concurrent HBV infection using Filipin III staining and ELISA. (D) Immunofluorescence observation of SREBP2 nuclear translocation in liver cancer, adjacent tissues, and normal liver tissues of patients with HBV infection (green arrows) (confocal 63x oil immersion lens). (E) More autolysosomes (red arrows) were observed in liver cancer tissues than in adjacent tissues through electron microscopy.*p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 3

Up‐regulation of SREBP2 and cholesterol levels and increased autophagy in HBV transgenic mice. (A) Immunohistochemical analysis of SREBP2 expression in liver tissues of wild‐type mice and HBV transgenic mice. (B) Western blotting was used to detect SREBP2 activation and nuclear translocation levels and autophagy‐related proteins in HBV transgenic mice and wild‐type mice. (C) Filipin III staining was used to detect cholesterol levels in liver tissues of HBV transgenic mice and wild‐type mice. (D) ELISA was used to detect cholesterol levels in the liver tissues of HBV transgenic mice and wild‐type mice. (E) Autophagosomes and autophagolysosomes (red arrows) were observed through transmission electron microscopy in hepatocytes of HBV transgenic mice and wild‐type mice.*p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 4

The expression and nucleation status of SREBP2 in cells. (A) Dual‐luciferase reporter gene assay to assess the direct activation of SREBP2 promoter by HBx; And construct different fragment truncations for the dual‐luciferase assay to identify the possible regulatory sites of HBx. (B) PCR detection of SREBF2 expression in HepG2 and Huh‐7 cells overexpressing HBx and HepG2.2.15 cells; (C) HepG2 and Huh‐7 cells overexpressing HBx, HepG2.2.15, and HBx‐silenced HepG2.2.15 cells were constructed, and the expression and activation of SREBP2 were detected using Western blotting technique; (D) FilipinIII and ELISA detection results of HepG2, Huh‐7, and HepG2.2.15 cells; (E) Immunofluorescence detection of the SREBP2 activated fragments and nucleus co‐localization; (F) Observe the effect of SREBP2 on cell proliferation through the EDU proliferation assay.*p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 5

HBx regulates cell autophagy through SREBP2. (A) The expression of autophagy markers in HepG2‐HBx, Huh7‐HBx, HepG2.2.15, and HepG2.2.15‐siHBx cells was measured by Western blotting techniques, as well as the changes in autophagy after SREBP2 knockdown or overexpression. (B) After HepG2 and HepG2.2.15 cells were seeded for 24 h, rapamycin (working concentration 30 uM) and bafilomycin A1 (working concentration 10 nM) were administered for 24 h according to the autophagy flux detection method. Similarly, after HepG2 cells were transiently transfected with HBx for 24 h, they were also treated with rapamycin and bafilomycin A1 for 24 h. (C) The autophagy of HepG2‐HBx, Huh7‐HBx, HepG2.2.15, and HepG2.2.15‐siHBx cells was assessed through the Puncta assay. (D) Autolysosomes (red arrows) in HepG2‐HBx, Huh7‐HBx, HepG2.2.15, and HepG2.2.15‐siHBx cells were observed through transmission electron microscopy.*p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 6

The proposed model shows the cross‐talk between lipid metabolism and HBV in HCC.