CRISPR/Cas9-mediated knockout of HBsAg inhibits proliferation and tumorigenicity of HBV-positive hepatocellular carcinoma cells

J Cell Biochem. 2018 Nov;119(10):8419-8431. doi: 10.1002/jcb.27050. Epub 2018 Jun 15.

Abstract

Chronic hepatitis B virus (HBV) infection remains the most common risk factor for hepatocellular carcinoma (HCC). High HBV surface antigen (HBsAg) levels are highly correlated with hepatocarcinogenesis and HBV-associated HCC development. However, the role and detailed mechanisms associated with HBsAg in HCC development remain elusive. In this study, we designed specific single guide RNAs (sgRNAs) targeting the open reading frames, preS1/preS2/S, of the HBV genome and established HBsAg knockout HCC cell lines using the CRISPR/Cas9 system. We showed that knockout of HBsAg in HCC cell lines decreased HBsAg expression and significantly attenuated HCC proliferation in vitro, as well as tumorigenicity in vivo. We also found that overexpression of HBsAg, including the large (LHBs), middle (MHBs), and small (SHBs) surface proteins promoted proliferation and tumor formation in HCC cells. Moreover, we demonstrated that knockout of HBsAg in HCC cells decreased interleukin (IL)-6 production and inhibited signal transducer and activator of transcription 3 (STAT3) signaling, while overexpression of HBsAg induced a substantial accumulation of pY-STAT3. Collectively, these results highlighted the tumorigenic role of HBsAg and implied that the IL-6-STAT3 pathway may be implicated in the HBsAg-mediated malignant potential of HBV-associated HCC.

Keywords: CRISPR/Cas9; HBV; HBsAg; HCC; IL-6; tumorigenicity.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • CRISPR-Cas Systems*
  • Carcinogenesis / metabolism*
  • Carcinoma, Hepatocellular / etiology
  • Carcinoma, Hepatocellular / pathology*
  • Cell Proliferation
  • Gene Knockout Techniques / methods
  • HEK293 Cells
  • Hep G2 Cells
  • Hepatitis B Surface Antigens / analysis
  • Hepatitis B Surface Antigens / genetics
  • Hepatitis B Surface Antigens / metabolism*
  • Hepatitis B virus / metabolism*
  • Hepatitis B, Chronic / complications
  • Humans
  • Interleukin-6 / analysis
  • Interleukin-6 / metabolism
  • Liver Neoplasms / etiology
  • Liver Neoplasms / pathology*
  • Mice, Nude
  • Transfection
  • Tumor Burden
  • Xenograft Model Antitumor Assays

Substances

  • Hepatitis B Surface Antigens
  • IL6 protein, human
  • Interleukin-6