EFNA 3 gene silencing inhibits on the migration and invasion of hepatocellular carcinoma cells induce by hypoxia
10.3781/j.issn.1000-7431.2020.11.111
- Author:
Jing CHEN
1
Author Information
1. State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji Hospital, Shanghai Jiao Tong University School of Medicine
- Publication Type:Journal Article
- Keywords:
Carcinoma;
Cell movement;
Competing endogenous RNA;
Hepatocellular;
Hypoxia
- From:
Tumor
2020;40(5):305-316
- CountryChina
- Language:Chinese
-
Abstract:
Objective: To investigate the effect of hypoxia on the expression of ephrin A3 (EFNA3) in hepatocellular carcinoma, and the effects of silencing EFNA3 gene expression on migration and invasion abilities of hepatocellular carcinoma cells induced by hypoxia. Methods: The hepatocellular carcinoma HCC-LY10 and MHCC-97L cells were cultured in hypoxic condition for 0, 12, 24 and 48 h, the expression level of EFNA3 mRNA and long non-coding RNA (LncRNA) EFNA3 were detected by real-time fluorescent quantitative PCR, the expression level of EFNA3 and hypoxia inducible factor-1α (HIF-1α) proteins were detected by Western blotting. The HCC-LY10 and MHCC-97L cells were transfected with microRNA (miR)-210-3pmimic and negative control (NC)-mimic by liposome, when cultured in normoxic and hypoxic condition for 48 h, the expression level of miR-210-3p, EFNA3 mRNA and LncRNA EFNA3 were detected by real-time fluorescent quantitative PCR, the expression level of EFNA3 and HIF-1α proteins were detected by Western blotting. The HCC-LY10 and MHCC-97L cells were infected with recombinant lentivirus vector carrying shHIF-1α and shHIF-2α targeting HIF-1α and HIF-2α genes (GV248-shHIF-1α-1, GV248-shHIF-1α-2, GV248-shHIF-2α-1 and GV248-shHIF-2α-2) and negative control plasmid (GV248-NC), when cultured in hypoxic condition for 48 h, the interference efficiency of HIF-1α and HIF-2α were detected by real-time fluorescent quantitative PCR, then the expression level of EFNA3 mRNA and LncRNA EFNA3 in HCC-LY10 and MHCC97L cells HIF-1α and HIF-2α silencing were detected by real-time fluorescent quantitative PCR. The HCC-LY10 and MHCC-97L cells were infected with recombinant lentivirus vector carrying shEFNA3 targeting EFNA3 genes (LVRU6GP-shEFNA3-1 and LVRU6GP-shEFNA3-2) and negative control plasmid (LVRU6GP-NC), the interference efficiency was analyzed by Western blotting. The effects of EFNA3 gene silencing on migration and invasion abilities of HCC-LY10 and MHCC-97L cells under hypoxic condition were evaluated by Transwell assays. Results: Under hypoxic condition, there was no significant change in EFNA3 mRNA level (P > 0.05), whereas EFNA3 protein level and LncRNA EFNA3 level increased significantly (all P < 0.01) in HCCLY10 and MHCC-97L cells. HCC-LY10 and MHCC-97L cells were transiently transfected miR-2103p-mimic, the miR-210-3p expression level was increased significantly (both P < 0.001). miR210-3p had little impact on the expression level of EFNA3 mRNA and LncRNA EFNA3 (both P > 0.05) and down-regulated the protein level of EFNA3 induced by hypoxia (both P < 0.05). Stably knocking-down HIF-1α and HIF-2α in HCC-LY10 and MHCC-97L cells under hypoxia, the mRNA expression level of HIF-1α and HIF-2α decreased (both P < 0.01). Knocking-down HIF-1α had no little impact on the mRNA level of EFNA3 (both P > 0.05), and decreased the level of LncRNA EFNA3 (both P < 0.001). Knocking-down HIF-2α had no little impact on the mRNA and LncRNA levels of EFNA3 (both P > 0.05). Stably knocking-down EFNA3 significantly inhibits the abilities of migration and invasion of HCC-LY10 and MHCC-97L cells induced by hypoxia (both P < 0.01). Conclusion: The expression level of LncRNA EFNA3 was up-regulated by HIF-1α under hypoxia competitively binding to miR-210-3p to result in the up-regulation of EFNA3 protein level. Knocking-down EFNA3 inhibits the migration and invasion abilities of hepatocellular carcinoma cells under hypoxic condition.
