CENPF Overexpression Induced by HBV Infection Facilitates the G1/S Cell Cycle Transition of Hepatocellular Carcinoma Cells via MYC Pathway

Saiping Qi,1– 5,* Donghu Zhou,1– 4,* Sisi Chen,1– 4,* Qin Ouyang,1– 4 Li-Na Wu,2,3,6 Hengcheng Tang,1– 4 Xiaojin Li,1– 4 Yanmeng Li,1– 4 Huaduan Zi,1– 4 Pingping He,1– 4 Xiaoming Wang,2– 4 Xiaojuan Ou,2– 4 Jiang Long,7 Li-Ying Sun,2,3,6 Jian Huang1– 4 1Laboratory of Molecular Biology, Beijing Institute of Clinical Medicine, Beijing Friendship Hospital, Capital Medical University, Beijing, People’s Republic of China; 2State Key Laboratory of Digestive Health, Beijing, People’s Republic of China; 3National Clinical Research Center for Digestive Diseases, Beijing, People’s Republic of China; 4Liver Research Center, Beijing Friendship Hospital, Capital Medical University, Beijing, People’s Republic of China; 5Department of Intensive Care Unit, Beijing Shijitan Hospital, Capital Medical University, Beijing, People’s Republic of China; 6Department of Critical Liver Diseases, Beijing Friendship Hospital, Capital Medical University, Beijing, People’s Republic of China; 7Beijing Minimally Invasive Oncology Medical Center of Traditional Chinese and Western Medicine, Dongzhimen Hospital, Beijing University of Chinese Medicine, Beijing, People’s Republic of China*These authors contributed equally to this workCorrespondence: Jian Huang, Laboratory of Molecular Biology, Beijing Institute of Clinical Medicine, Beijing Friendship Hospital, Capital Medical University, Beijing, 100050, People’s Republic of China, Email [email protected] Li-Ying Sun, Department of Critical Liver Diseases, Beijing Friendship Hospital, Capital Medical University, Beijing, 101125, People’s Republic of China, Email [email protected]: Centromere protein F (CENPF), a mitosis-related protein, is overexpressed in hepatocellular carcinoma (HCC) and has emerged as a promising biomarker for early HCC. However, the role of hepatitis B virus (HBV) infection on CENPF overexpression in HCC remains unknown. Moreover, with ultra-large molecular weight of 358kDa of CENPF, no study has directly explored its carcinogenicity with an overexpression model.Materials and Methods: The relationship among HBV infection, CENPF amplification and CENPF overexpression was investigated in HCC tissues. HBV X protein (HBx) transient overexpression cell models was constructed to explore its effect on CENPF expression. CENPF was upregulated and downregulated to analyze its functions in vitro and in vivo. Specifically, a CRISPR/dCas9 system was applied to construct the CENPF overexpression model.Results: A high frequency of CENPF amplification (36.21%, 21/58) was identified in HCC tissues, predominantly in HBV-associated cases (90.48%, 19/21), and CENPF amplification correlated with CENPF overexpression. The HBx enhanced CENPF expression in HBx transfected HCC cells. In addition, CENPF knockdown cell models showed inhibition of HCC proliferation both in vitro and in vivo. Notably, as a cell cycle protein with high constitutive expression in G2/M phase, CENPF overexpression cell models also showed inhibitory effects, probably due to the toxic effect of excessive CENPF expression on G2/M transition. However, in both CENPF downregulation and overexpression models, cell cycle assays showed CENPF promoted G1/S transition in HCC cells. RNA-seq showed that CENPF overexpression activated the MYC pathway, thereby promoting G1/S transition. Rescue experiment indicated that the MYC pathway inhibitor 10058-F4 counteracted the G1/S transition induced by CENPF overexpression in HCC cells.Conclusion: HBV infection was associated with upregulated CENPF expression in HCC and CENPF overexpression might facilitate G1/S transition of HCC cells via the MYC pathway.Keywords: hepatocellular carcinoma, HBV infection, CENPF amplification, CENPF overexpression, G1/S cell cycle transition