Genome-wide loss-of-function genetic screen identifies INSIG2 as the vulnerability of hepatitis B virus-integrated hepatoma cells

Abstract

There are approximately 250 million people chronically infected with hepatitis B virus (HBV) worldwide. Although HBV is often integrated into the host genome and promotes hepatocarcinogenesis, vulnerability of HBV integration in liver cancer cells has not been clarified. The aim of our study is to identify vulnerability factors for HBV-associated hepatocarcinoma. Loss-of-function screening was undertaken in HepG2 and HBV-integrated HepG2.2.15 cells expressing SpCas9 using a pooled genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) library. Genes whose guide RNA (gRNA) abundance significantly decreased in HepG2.2.15 cells but not in HepG2 cells were extracted using the MAGeCK algorithm. We identified four genes (BCL2L1, VPS37A, INSIG2, and CFLAR) that showed significant reductions of gRNA abundance and thus potentially involved in the vulnerability of HBV-integrated cancer cells. Among them, siRNA-mediated mRNA inhibition or CRISPR-mediated genetic deletion of INSIG2 significantly impaired cell proliferation in HepG2.2.15 cells but not in HepG2 cells. Its inhibitory effect was alleviated by cotransfection of siRNAs targeting HBV. INSIG2 inhibition suppressed the pathways related to cell cycle and DNA replication, downregulated cyclin-dependent kinase 2 (CDK2) levels, and delayed the G₁ -to-S transition in HepG2.2.15 cells. CDK2 inhibitor suppressed cell cycle progression in HepG2.2.15 cells and INSIG2 inhibition did not suppress cell proliferation in the presence of CDK2 inhibitor. In conclusion, INSIG2 inhibition induced cell cycle arrest in HBV-integrated hepatoma cells in a CDK2-dependent manner, and thus INSIG2 might be a vulnerability factor for HBV-associated liver cancer.

Keywords: CDK2; CRISPR/Cas; HBV; liver cancer; pooled library screen.

FIGURE 1

Pooled clustered regularly interspaced short palindromic repeat (CRISPR) library screen Identifies four gene candidates related to the vulnerability of hepatitis B virus (HBV)‐integrated liver cancer cells. (A) Western blot (WB) analysis of Cas9 protein levels in the HepG2 and HepG2.2.15 cell lines. Clone, Cas9‐transduced monoclonal cells; Mock, Cas9‐nontransduced cells. (B) MAGeCK analysis showed significantly depleted (false discover rate <0.05) genes compared to the beginning of the culture both in HepG2 cells and HepG2.2.15 cells. (C) Negative log10 p value for each gene. Horizontal axis shows depletion in HepG2.2.15 cells compared to HepG2 cells at day 14. Vertical axis shows depletion in HepG2.2.15 cells at day 14 compared to day 2. (D) Log2 fold change values of each guide RNA (gRNA) targeting gene (BCL2L1, INSIG2, VPS37A, and CFLAR) in HepG2.2.15 cells compared to those in HepG2 cells. Note that the fold change of gRNA6 in BCL2L1, INSIG2, and VPS37A were not calculated. Abbreviations: ACTB, β‐actin.

FIGURE 2

Deletion of BCL2L1 and INSIG2 specifically reduces cell proliferation in hepatitis B virus‐integrated liver cancer cells. (A) Cell viability was measured by WST‐1 assays 4 days after knockdown with siRNA (ratio to negative control [NC], *p < 0.05). (B) Western blot (WB) analysis of BCL2L1 in HepG2‐Cas9 cells and HepG2.2.15 cells after transfection with gBCL2L1. (C) WB analysis of INSIG2 in HepG2‐Cas9 cells and HepG2.2.15 cells after transfection with gINSIG2. (D) Cell viability in HepG2/HepG2.2.15‐gBCL2L1‐Knock Out (KO) cells at 6 days after seeding (ratio to NC, *p < 0.05). (E) Cell viability in HepG2/HepG2.2.15‐gINSIG2‐KO cells at 6 days after seeding (ratio to NC, *p < 0.05). Abbreviations: ACTB, β‐actin; g1/g2/g3, gRNA‐1/gRNA‐2/gRNA‐3.

FIGURE 3

Vulnerability induced by INSIG2 inhibition in hepatitis B virus (HBV)‐integrated liver cancer cells is partly dependent on the presence of HBV. (A) Supernatant hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) levels, HBV core (HBc) protein levels, and intracellular pregenomic RNA (pgRNA) levels in HepG2.2.15 cells 6 days after transfection with HBV siRNA. (*p < 0.05). (B) Cell viability was measured by WST‐1 assays 4 days after transfection with siBCL2L1 and/or HBV siRNA (ratio to negative control [NC], *p < 0.05). (C) Cell viability was measured by WST‐1 assays 4 days after transfection with siINSIG2 and/or HBV siRNA (ratio to NC, *p < 0.05). Abbreviations: ACTB, β‐actin; ns, not significant.

FIGURE 4

INSIG2 inhibition delays cell cycle, leading to a decrease in cell proliferation in hepatitis B virus‐integrated liver cancer cells. (A) Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis results. Horizontal axis indicates the negative log10 p value for each term. (B) To synchronize G₂/M, cells were treated with nocodazole (400 ng/mL) after knockdown (KD) with control siRNA (siNC)/INSIG2. Then, HepG2/HepG2.2.15 cells were assessed by flow cytometry. (C‐F) To synchronize G₁/S, cells were treated by double thymidine block (2 mM). Then, cells were assessed by flow cytometry immediately and 8 h after release. (C) HepG2 cells after KD with siNC/INSIG2. (D) HepG2 cell population ratio at each stage of cell cycle. (E) HepG2.2.15 cells after KD with siNC/INSIG2. (F) HepG2.2.15 cell population ratio at each stage of cell cycle. Abbreviations: PI, propidium iodide; PPAR, peroxisome proliferator‐activated receptor.

FIGURE 5

Cyclin‐dependent kinase 2 (CDK2) is involved in the vulnerability caused by INSIG2 inhibition in hepatitis B virus‐integrated liver cancer cells. (A) Western blot analysis of G₁/S cell cycle factors in HepG2‐Cas9 cells and HepG2.2.15 cells after transfection with control siRNA (siNC)/siINSIG2. (B) Alteration of cell cycle by CVT‐313 treatment. After 24 h of treatment (left) with no CVT‐313 or (right) with CVT‐313 (5 μM), CDK2 inhibition by CVT‐313 resulted in cell cycle arrest at the G₁/S transition. (C) Cell population ratio at each stage of cell cycle is shown. (D) Cell viability was measured by WST‐1 assays 4 days after CVT‐313 (5uM) treatment in HepG2.2.15 cells (ratio to no treatment [no treat], *p < 0.05). (E) Cell viability was measured by WST‐1 assays 4 days after CVT‐313 treatment and transfection with siNC/siINSIG2 (ratio to no treat). Abbreviations: ACTB, β‐actin; cycA/D/E, cyclinA/D/E; ns, not significant; PI, propidium iodide.

FIGURE 6

INSIG2 affects cyclin‐dependent kinase 2 (CDK2) through hepatitis B X protein (HBx). (A) Western blot (WB) analysis of extracts from cells transfected with Myc‐INSIG2 and/or FLAG‐HBx, using MYC and FLAG tag Abs. (B) After cells were transfected with MYC‐INSIG2 and/or FLAG‐HBx, immunoprecipitation with FLAG tag Ab was carried out. WB analysis using Myc Tag Ab. (C) Schematic diagram of this study. INSIG2 and CDK2 may be molecularly bound through HBx in hepatitis B virus (HBV)‐integrated cancer cells, creating the dependency of INSIG2 on their cell proliferation. Abbreviations: ACTB, β‐actin; HCC, hepatocellular carcinoma.