Effect of 5-Aza-dC on FHIT gene expression in hepatocellular carcinoma cell line HepG2

Abstract

FHIT gene methylation leads to down-regulation of its expression in hepatocellular carcinoma (HCC) cells. This study was to detect the expression of FHIT mRNA and protein in HCC cell line HepG2 after treatment of methylase inhibitor 5-Aza-2'-deoxycytidine (5-Aza-dC), and observe the effect of 5-Aza-dC on the proliferation of HepG2 cells. HepG2 cells were treated with 5-Aza-dC. Methylation of FHIT in HepG2 cells was detected by methylation-specific polymerase chain reaction (MSP). FHIT mRNA expression was detected by reverse transcription-polymerase chain reaction (RT-PCR). FHIT protein expression was detected by immunohistochemistry and Western blot. Cell proliferation was detected by MTT assay. Before treatment of 5-Aza-dC, FHIT gene methylation was detected in HepG2 cells, while no FHIT mRNA and protein expression was detected. The hypermethylation of FHIT gene in HepG2 cells was effectively reversed after treatment of 5-Aza-dC. When HepG2 cells were treated with 1.0, 2.0, and 4.0 micromol/L of 5-Aza-dC for 48 h, the mRNA levels of FHIT were 0.80+/-0.32, 1.41+/-0.54, and 1.51+/-0.61, respectively; the protein levels of FHIT were 0.33+/-0.20, 1.00+/-0.26, and 1.12+/-0.38, respectively. Cell proliferation was significantly inhibited after being treated with 5-Aza-dC. 5-Aza-dC can reverse the abnormal methylation of FHIT gene, activate the silenced gene and induce FHIT mRNA and protein expression in HepG2 cells.