Effects of deoxyribonucleic acid methylation on the expression of hepatocyte nuclear factor-4α
10.3760/cma.j.issn.0254-1432.2013.09.010
- VernacularTitle:脱氧核糖核酸甲基化对肝细胞核因子4α表达的影响
- Author:
Jingxian HU
;
Xin ZHANG
;
Weifen XIE
- Publication Type:Journal Article
- Keywords:
Carcinoma,hepatocellular;
Hepatocyte nuclear factor 4;
DNA methylation;
Transforming growth factor beta 1;
Deoxycytidine
- From:
Chinese Journal of Digestion
2013;33(9):621-625
- CountryChina
- Language:Chinese
-
Abstract:
Objective To investigate the effect of deoxyribonucleic acid (DNA) methylation on the expression of hepatocyte nuclear factor-4α (HNF4c) and its role in the expression of HNF4α regulated by transforming growth factor-β1 (TGF-β1).Methods The expression of HNF4αP1 mRNA in six human hepatoma cell lines (HepG2,Huh-7,Hep3B,SMMC-7721,BEL-7405 and FOCUS),20 hepatic carcinoma specimens and corresponding adjacent tissues was detected by real-time reverse transcription polymerse chain reaction (real-time RT-PCR).The methylation status of the promoter region of HNF4αP1 in six human hepatoma cell lines was examined by bisulfite sequencing PCR (BSP).FOCUS cells were treated with 5-aza-2'-deoxycytidine (5-AZA-CdR) and then the methylation status of the promoter region of HNF4αP1 was examined by BSP.The expression of HNF4αP1 mRNA was detected by real-time RT-PCR.The six human hepatoma cell lines were treated with TGFβ1 and the expression of HNF4αP1 mRNA was detected by real-time RT-PCR.FOCUS cells were cotreated with 5-AZA-CdR,TGF-β1 and 5-AZA-CdR.The expression of HNF4αP1 mRNA was detected by real-time RT-PCR,and t test was performed for statistical analysis.Results Among 20 human hepatic carcinoma specimens and corresponding adjacent tissues,the expression of HNF4αP1 mRNA of 13 human hepatic carcinoma specimens was lower than that of corresponding adjacent tissues (t=2.350,P＜0.05).The relative quantity of the expression of HNF4αP1 mRNA was higher in Hep3B,HepG2 and Huh-7 cells,whereas that in SMMC-7721,BEL-7405 and FOCUS cells was lower.The methylation of the promoter region of HNF4αP1 in HepG2,Huh-7 and Hep3B was lower,but that in SMMC-7721,BEL-7405 and FOCUS was higher.Along with the increasing of the concentration of 5-AZA-CdR (0,0.1,1.0 and 2.5 μmol/L),the degree of the methylation of the promoter region of HNF4αP1 in FOCUS cells gradually decreased (61％,46％,32％ and 27％),and however the relative quantity of the expression of HNF4αP1 mRNA gradually increased ((9.661 ± 0.336)×10-7,(2.001±0.432)×10-6,(3.689±0.714)×10-6and (4.732±2.451)×10-6).After stimulated with TGF-β1,the relative quantity of the expression of HNF4αP1 mRNA was downregulated in HepG2,Huh-7 and Hep3B cells in which the methylation of the promoter region was low (t=12.994,8.441,and 9.032,all P＜0.01).There was no significant difference in the relative quantity of the expression of HNF4αP1 mRNA in SMMC-7721,BEL-7405 and FOCUS cells in which the methylation of the promoter region was high (all P ＞ 0.05).The relative quantity of the expression of HNF4αP1 mRNA in 5-AZA-CdR treated FOCUS cells ((4.972±0.035) × 10-6) was higher than that of control group ((1.411 ± 0.104) × 10-6) and the difference was statistically significant (t=13.212,P＜0.01).The relative quantity of the expression of HNF4αP1 mRNA in FOCUS cells co-treated with 5-AZA-CdR and TGF-β1 was lower than that in cells treated with 5-AZA-CdR alone and the difference was statistically significant ((1.181 ± 0.132) × 10-6 vs (4.972 ± 0.035) × 10-6,t=13.873,P＜0.01).Conclusions The expression of HNF4αP1 is down-regulated in hepatic carcinoma tissues.DNA methylation may regulate the expression of HNF4αP1 in hepatoma cells.The methylation of HNF4αP1 promoter region inhibits the regulating function of TGF-β1 in the expression of HNF4αP1.
