Downregulation of demethylase FTO inhibits proliferation of human liver cancer cell line HepG2

Abstract

Objective To explore the mechanism of the demethylase fat mass and obesity associatal(FTO) gene on the proliferation of human liver cancer cell line HepG2. Methods HepG2 cells were divided into the control group, FTO group (transfected with FTO over-expression plasmid), si-FTO group (transfected with si-FTO)and si-FTO+si-FoxO1 group (simultaneously transfected with si-FTO and si-FoxO1). The expression of FTO in cells was detected by RT-qPCR. Cell proliferation, invasion and apoptosis were examined using CCK-8 assay, Transwell chamber assay and flow cytometry, respectively. Dot blot assay was performed to measure m6A methylation , and protein expression of FoxO1 in cells was detected by Western blot. Results Analysis of overall survival in liver cancer patients using The Cancer Genome Atlas (TCGA) showed higher expression of FTO associated with shorter survival (P＜0.05). Compared with normal liver cells HL7702, FTO relative expression was significantly increased in human liver cancer cells HepG2 (P＜0.05).The relative expression of FTO in si-FTO group cells was lower than that in the control group, while the relative expression of FTO in FTO group was higher than that in the control group (P＜0.05). The relative expression of FTO in the si-FTO+si-FoxO1 group was higher than that in the si-FTO group(P＜0.05). Compared with the Control group, cell viability, count of invasive cells, relative level of m6A were significantly decreased, while apoptosis rate and protein expression of FoxO1 were significantly increased in the si-FTO group(P＜0.05). Cell viability, count of invasive cells, and relative expression level of m6A were significantly higher, while apoptosis rate and protein expression of FoxO1 were significantly lower in the FTO group compared to the Control group (P＜0.05). Compared with the si-FTO group, cell viability, invasive cells and relative level of m6A were significantly increased, while apoptosis rate and protein expression of FoxO1 were significantly decreased in the si-FTO+si-FoxO1 group(P＜0.05). Conclusion High expression of FTO is associated with poor clinical prognosis. Knockdown of the demethylase FTO gene inhibits proliferation and invasion of liver cancer cells and induces their apoptosis. The mechanism is potentially related to the regulation of FoxO1.

Keywords

• demethylase|fat mass and obesity associated(fto) protein|forkhead box protein 1(foxo1)|hepatocellular carcinoma|apoptosis