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

Silencing of Tumor Necrosis Factor Receptor 1 by siRNA in EC109 Cells Affects Cell Proliferation and Apoptosis

Tumor necrosis factor receptor 1 (TNFR1) is a membrane receptor able to bind TNF-α or TNF-β. TNFR1 can suppress apoptosis by activating the NF-κB or JNK/SAPK signal transduction pathway, or it can induce apoptosis through a series of caspase cascade reactions; the particular effect may depend on the cell line. In the present study, we first showed that TNFR1 is expressed at both the gene and protein levels in the esophageal carcinoma cell line EC109. Then, by applying a specific siRNA, we silenced the expression of TNFR1; this resulted in a significant time-dependent promotion of cell proliferation and downregulation of the apoptotic rate. These results suggest that TNFR1 is strongly expressed in the EC109 cell line and that it may play an apoptosis-mediating role, which may be suppressed by highly activated NF-κB

miR-195-5p Regulates Hair Follicle Inductivity of Dermal Papilla Cells by Suppressing Wnt/β-Catenin Activation

Dermal papilla (DP) cells play a vital role in hair follicle (HF) development and postnatal hair cycling. However, the abilities are lost on further culture. Recent studies have demonstrated significant influences of posttranscriptional regulation by microRNA (miRNA) on HF development. The current study aims to investigate how miRNAs regulate Wnt/β-catenin to control HF inductivity of DP cells by performing microarray analysis in early- and late-passage DP cells and transfecting with miRNAs inhibitor or mimic. Results showed early-passage DP cells strongly expressed miRNAs related to inhibition of noncanonical Wnt pathways. In late-passage DP cells, miRNAs capable of inhibiting the canonical Wnt/β-catenin pathway were upregulated, in addition to the miRNAs targeting the noncanonical Wnt pathway. Moreover, we verified that β-catenin expression was downregulated by miR-195-5p overexpression in dose manner. Meanwhile LRP6 expression was downregulated in both protein and mRNA as well as the genes involved in the hair inductivity of DP cells. These results suggest that the appearance of miRNAs that suppress the Wnt/β-catenin pathway may be responsible for the loss of ability of DP cells in culture and miR-195-5p is the potential key factor involved in regulating HF inductivity of DP cells

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

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

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