Downregulation and pro-apoptotic effect of hypoxia-inducible factor 2 alpha in hepatocellular carcinoma.

Sheng-Li Yang,George G Chen,Jia Fan,Li-Ping Liu,Paul B.S Lai,Jian-Wei Ren,Leilei Niu,Xing-Rong Yang,Yun-Fan Sun

doi:10.18632/oncotarget.8952

Abstract

The role of HIF-2α in hepatocellular carcinoma (HCC) is unclear. The aim of the present study was to investigate the expression pattern and role of HIF-2α in HCC patients. Immunohistochemical staining and western blotting analyses were applied to detect the protein level of HIF-2α in 206 paired HCC and peritumoral tissues. Kaplan-Meier survival and Cox proportional hazard regression analyses were performed to identify risk factors for overall survival and recurrence-free survival in these patients. The function of HIF-2α was studied in HCC cells and in vivo models. We found that the protein levels of HIF-2α in HCC tissues were lower than in peritumoral tissues, and were negatively correlated with tumor size (P < 0.05). Kaplan-Meier survival and univariate analysis revealed that HCC patients with high HIF-2α protein levels had longer overall survival (P < 0.05). Over-expression of HIF-2α induced apoptosis in HCC cells and increased the levels of pro-apoptotic proteins, Bak, ZBP-89 and PDCD4, whereas the inhibition of HIF-2α expression achieved opposite results. The findings were confirmed in a mouse HCC xenograft model. In conclusion, our study revealed that HIF-2α was decreased and played an anti-tumorigenic role in HCC.

Highlights

The human HCC cell line HepG2 was purchased from American Type Culture Collection (Rockville, MD)
The polymerase chain reaction (PCR) fragments of four promoter were inserted into luciferase reporter vector pGL3-Basic (Promega, Madison, WI, USA) digested by SacI/XhoI (Takara Biotechnology Co., Ltd., Dalian, China)
After 48 h, the cells were co-transfected with promoter plasmid (Bak or ZBP-89 or PDCD4 or pGL3Basic), pcDNA3.1 or pcDNA3.1-HIF-2α, pRL-TK Renilla Luciferase Reporter (Promega), pshRNA-HIF2α or a negative control with aid of Lipofectamine 2000 (Invitrogen) according to the manufacturers’ instructions

Summary

Introduction

The human HCC cell line HepG2 was purchased from American Type Culture Collection (Rockville, MD). The human HCC cell lines (Hep3B, and Bel-7404) were obtained from the Institute of Cell Biology (SIBS, CAS, Shanghai, China). The cell was cultured in the atmosphere with 5% CO2 at 37°C. For mimicking the hypoxic conditions, the cell was cultured in an atmosphere with 1% O2, 5% CO2, and 94% N2 at 37°C (Incubator: Galaxy R incubator, Model No: 170-300, RS Biotech, UK).

Results

Conclusion