Human Papillomavirus Type 18 E6 and E7 Genes Integrate into Human Hepatoma Derived Cell Line Hep G2

Background and Objectives: Human papillomaviruses have been linked causally to some human cancers such as cervical carcinoma, but there is very little research addressing the effect of HPV infection on human liver cells. We chose the human hepatoma derived cell line Hep G2 to investigate whether HPV gene integration took place in liver cells as well. Methods: We applied PCR to detect the possible integration of HPV genes in Hep G2 cells. We also investigated the expression of the integrated E6 and E7 genes by using RT-PCR and Western blotting. Then, we silenced E6 and E7 expression and checked the cell proliferation and apoptosis in Hep G2 cells. Furthermore, we analyzed the potential genes involved in cell cycle and apoptosis regulatory pathways. Finally, we used in situ hybridization to detect HPV 16/18 in hepatocellular carcinoma samples. Results: Hep G2 cell line contains integrated HPV 18 DNA, leading to the expression of the E6 and E7 oncogenic proteins. Knockdown of the E7 and E6 genes expression reduced cell proliferation, caused the cell cycle arrest at the S phase, and increased apoptosis. The human cell cycle and apoptosis real-time PCR arrays analysis demonstrated E6 and E7-mediated regulation of some genes such as Cyclin H, UBA1, E2F4, p53, p107, FASLG, NOL3 and CASP14. HPV16/18 was found in only 9% (9/100) of patients with hepatocellular carcinoma. Conclusion: Our investigations showed that HPV 18 E6 and E7 genes can be integrated into the Hep G2, and we observed a low prevalence of HPV 16/18 in hepatocellular carcinoma samples. However, the precise risk of HPV as causative agent of hepatocellular carcinoma needs further study

mRNA expression of E7-related genes in Hep G2 cell after the silencing of HPV 18 E7 in RT² Profiler™ Human Apoptosis PCR Array.

<p>In 84 E7-related genes, 21 genes mRNA transcript were altered markedly compared with the negative control. Standards of eligibility: folds up- or down-regulation >2.0 and <i>p</i><0.05; n = 3.</p

Transfection with E7-siRNA induced apoptosis.

<p>(A, B, C and D) The percent of apoptotic Hep G2 cells was measured using the Annexin V assay at 24 h, 48 h and 72 h after transfection, and then the stained cells were analyzed through flow cytometry. (E) The results showed that 7.26%±0.29%, 22.03%±0.23% and 19.20%±0.78% in siRNA E7 transfected Hep G2 cells after 24 h, 48 h and 72 h underwent total apoptosis compared with only 5.25%±0.76% in NC siRNA E7 transfected cells (p<0.05).</p

siRNA targeting HPV 18 E7 sites and sequences.

<p>siRNA targeting HPV 18 E7 sites and sequences.</p

These photomicrographs show in situ hybridization results for human papillomavirus positive hepatocellular carcinoma.

<p>(A) Hep G2 cells were with the punctate signal pattern of HPV DNA. (B) HeLa cells were served as positive control. (C) Hepatocellular carcinoma was with diffuse signal pattern of HPV staining. (D) No signal was found in hepatoma carcinoma cells of this HPV- negative specimen.</p

Inhibition of HPV 18 E7 gene inhibited cell growth in Hep G2.

<p>(A) Hep G2 cells transfected with E7-siRNA or control siRNA were evaluated in EdU assays at 0 h, 12 h, 24 h, 36 h, 48 h, 60 h and 72 h after transfection. After transfection of Hep G2 cells with E7-siRNA, a time-dependent reduction of cell proliferation was observed at 48 h, 60 h and 72 h. (B) At 48 h, 60 h and 72 h following E7-siRNA transfection, the percents of S-phase cells were 9.39%±3.55%, 17.29%±5.85% and 30.87%±4.26% compared with 18.74%±6.66%, 24.03%±5.35% and 41.97%±8.73% in NC-siRNA transfected cells (p<0.05).</p

mRNA expression of E7-related genes in Hep G2 cells after the silencing of HPV 18 E7 through RT² Profiler™ Human Cell Cycle PCR Array.

<p>In 84 E7-related genes, 19 genes mRNA transcript were altered markedly compared with the negative control. Standards of eligibility: folds up- or down-regulation >2.0 and <i>p</i><0.05; n = 3.</p

Hep G2 cells with integrated HPV 18 DNA expressed E6 and E7 mRNAs and proteins.

<p>(A) PCR amplification of HPV 18 E6E7 gene was assessed in samples from Hep G2, EC109, and K562 cells. Then an amplified fragment of 847 bp was present in both Hep G2 and EC109 cells (HPV 18 positive), but absent in K562 cells (HPV 18 negative). (B) The expression of HPV 18 E6 and E7 mRNA was detected by RT-PCR. The expected fragments of E6 (196 bp) and E7 (332 bp) were present in both Hep G2 and EC109 cells, but not in K562 cells. (C) Western blotting showed the expression of the HPV 18 E6 and E7 proteins. EC109 and K562 were used as controls. β-actin was used as an internal control.</p

Immunohistochemistry demonstrated that the Hep G2 cell line was of characteristics typical of liver cells.

<p>Immunohistochemical evaluation of Hep G2 cells with anti-human hepatocyte antibody indicated that the Hep G2 cell cytoplasm (A), but not HeLa cells (B), exhibited typical hepatocyte antigens.</p