CAJ | 학술논문

目的探讨DNA甲基化对肝细胞核因子4α(HNF4α)表达的影响,及其在转化生长因子-β1 (TGF-β1)调节HNF4α表达过程中所起的作用.方法用实时RT-PCR方法检测6种人肝癌细胞株(HepG2、Huh-7、Hep3B、SMMC-7721、BEL-7405、FOCUS)、20份人肝癌和相应癌旁组织中HNF4αP1 mRNA的表达.用亚硫酸盐测序法(BSP法)检测6种人肝癌细胞株中HNF4αP1启动子区甲基化程度.用5-脱氧杂氮胞苷(5-AZA-CdR)处理FOCUS细胞株,用BSP法检测HNF4αP1启动子区甲基化程度,用实时RT-PCR法检测HNF4αP1 mRNA的表达.用TGF-β1处理6种人肝癌细胞株,用实时RT-PCR法检测HNF4αP1 mRNA的表达.分别用5-AZA-CdR、TGF-β1、5-AZA-CdR联合TGF-β1处理FOCUS细胞株,用实时RT-PCR法检测HNF4αP1 mRNA的表达.采用f检验进行统计学分析.结果在20份人肝癌和癌旁组织中,13份肝癌组织的HNF4αP1 mRNA相对表达量低于癌旁组织(f=2.350,P＜0.05).HepG2、Huh-7、Hep3B中的HNF4αP1 mRNA相对表达量较高,SMMC-7721、BEL-7405、FOCUS中则相对较低.HepG2、Huh-7、Hep3B中HNF4αP1启动子区甲基化程度较低,SMMC-7721、BEL-7405、FOCUS中则较高.随着5-AZA-CdR浓度上升(分别为0、0.1、1.0、2.5 μmol/L),FOCUS的HNF4αP1启动子甲基化程度逐渐降低(分别为61％、46％、32％、27％),而HNF4αP1 mRNA相对表达量则逐渐增加(分别为[(9.661±0.336)×10-7、(2.001±0.432)×10-6、(3.689±0.714)×10-6、(4.732±2.451)×10-6].经TGF-β1刺激后,启动子区甲基化程度较低的HepG2、Huh-7、Hep3B的HNF4αP1 mRNA相对表达量下调(t=12.994、8.441、9.032,P均＜0.01),启动子区甲基化程度较高的SMMC-7721、BEL-7405、FOCUS的HNF4αP1 mRNA相对表达量变化差异均无统计学意义(P均＞0.05).经5-AZA-CdR处理的FOCUS细胞株的HNF4αP1 mRNA相对表达量[(4.972±0.035)×10-6]高于空白对照组[(1.411±0.104)×10-6],差异有统计学意义(t=13.212,P＜0.01).经5-AZA-CdR联合TGF-β1处理的FOCUS细胞株的HNF4αP1 mRNA相对表达量[(1.181±0.132)×10-6]低于单用5-AZA-CdR处理组[(4.972±0.035)×10-6],差异有统计学意义(t=13.873,P＜0.01).结论肝癌组织中HNF4αP1表达水平下调.DNA甲基化可调控HNF4αP1在人肝癌细胞株中的表达.HNF4αP1启动子区甲基化可抑制TGF-β1对HNF4αP1表达的调节作用. 目的探討DNA甲基化對肝細胞覈因子4α(HNF4α)錶達的影響,及其在轉化生長因子-β1 (TGF-β1)調節HNF4α錶達過程中所起的作用.方法用實時RT-PCR方法檢測6種人肝癌細胞株(HepG2、Huh-7、Hep3B、SMMC-7721、BEL-7405、FOCUS)、20份人肝癌和相應癌徬組織中HNF4αP1 mRNA的錶達.用亞硫痠鹽測序法(BSP法)檢測6種人肝癌細胞株中HNF4αP1啟動子區甲基化程度.用5-脫氧雜氮胞苷(5-AZA-CdR)處理FOCUS細胞株,用BSP法檢測HNF4αP1啟動子區甲基化程度,用實時RT-PCR法檢測HNF4αP1 mRNA的錶達.用TGF-β1處理6種人肝癌細胞株,用實時RT-PCR法檢測HNF4αP1 mRNA的錶達.分彆用5-AZA-CdR、TGF-β1、5-AZA-CdR聯閤TGF-β1處理FOCUS細胞株,用實時RT-PCR法檢測HNF4αP1 mRNA的錶達.採用f檢驗進行統計學分析.結果在20份人肝癌和癌徬組織中,13份肝癌組織的HNF4αP1 mRNA相對錶達量低于癌徬組織(f=2.350,P＜0.05).HepG2、Huh-7、Hep3B中的HNF4αP1 mRNA相對錶達量較高,SMMC-7721、BEL-7405、FOCUS中則相對較低.HepG2、Huh-7、Hep3B中HNF4αP1啟動子區甲基化程度較低,SMMC-7721、BEL-7405、FOCUS中則較高.隨著5-AZA-CdR濃度上升(分彆為0、0.1、1.0、2.5 μmol/L),FOCUS的HNF4αP1啟動子甲基化程度逐漸降低(分彆為61％、46％、32％、27％),而HNF4αP1 mRNA相對錶達量則逐漸增加(分彆為[(9.661±0.336)×10-7、(2.001±0.432)×10-6、(3.689±0.714)×10-6、(4.732±2.451)×10-6].經TGF-β1刺激後,啟動子區甲基化程度較低的HepG2、Huh-7、Hep3B的HNF4αP1 mRNA相對錶達量下調(t=12.994、8.441、9.032,P均＜0.01),啟動子區甲基化程度較高的SMMC-7721、BEL-7405、FOCUS的HNF4αP1 mRNA相對錶達量變化差異均無統計學意義(P均＞0.05).經5-AZA-CdR處理的FOCUS細胞株的HNF4αP1 mRNA相對錶達量[(4.972±0.035)×10-6]高于空白對照組[(1.411±0.104)×10-6],差異有統計學意義(t=13.212,P＜0.01).經5-AZA-CdR聯閤TGF-β1處理的FOCUS細胞株的HNF4αP1 mRNA相對錶達量[(1.181±0.132)×10-6]低于單用5-AZA-CdR處理組[(4.972±0.035)×10-6],差異有統計學意義(t=13.873,P＜0.01).結論肝癌組織中HNF4αP1錶達水平下調.DNA甲基化可調控HNF4αP1在人肝癌細胞株中的錶達.HNF4αP1啟動子區甲基化可抑製TGF-β1對HNF4αP1錶達的調節作用.
목적 탐토DNA갑기화대간세포핵인자4α(HNF4α)표체적영향,급기재전화생장인자-β1 (TGF-β1)조절HNF4α표체과정중소기적작용.방법 용실시RT-PCR방법검측6충인간암세포주(HepG2、Huh-7、Hep3B、SMMC-7721、BEL-7405、FOCUS)、20빈인간암화상응암방조직중HNF4αP1 mRNA적표체.용아류산염측서법(BSP법)검측6충인간암세포주중HNF4αP1계동자구갑기화정도.용5-탈양잡담포감(5-AZA-CdR)처리FOCUS세포주,용BSP법검측HNF4αP1계동자구갑기화정도,용실시RT-PCR법검측HNF4αP1 mRNA적표체.용TGF-β1처리6충인간암세포주,용실시RT-PCR법검측HNF4αP1 mRNA적표체.분별용5-AZA-CdR、TGF-β1、5-AZA-CdR연합TGF-β1처리FOCUS세포주,용실시RT-PCR법검측HNF4αP1 mRNA적표체.채용f검험진행통계학분석.결과 재20빈인간암화암방조직중,13빈간암조직적HNF4αP1 mRNA상대표체량저우암방조직(f=2.350,P＜0.05).HepG2、Huh-7、Hep3B중적HNF4αP1 mRNA상대표체량교고,SMMC-7721、BEL-7405、FOCUS중칙상대교저.HepG2、Huh-7、Hep3B중HNF4αP1계동자구갑기화정도교저,SMMC-7721、BEL-7405、FOCUS중칙교고.수착5-AZA-CdR농도상승(분별위0、0.1、1.0、2.5 μmol/L),FOCUS적HNF4αP1계동자갑기화정도축점강저(분별위61％、46％、32％、27％),이HNF4αP1 mRNA상대표체량칙축점증가(분별위[(9.661±0.336)×10-7、(2.001±0.432)×10-6、(3.689±0.714)×10-6、(4.732±2.451)×10-6].경TGF-β1자격후,계동자구갑기화정도교저적HepG2、Huh-7、Hep3B적HNF4αP1 mRNA상대표체량하조(t=12.994、8.441、9.032,P균＜0.01),계동자구갑기화정도교고적SMMC-7721、BEL-7405、FOCUS적HNF4αP1 mRNA상대표체량변화차이균무통계학의의(P균＞0.05).경5-AZA-CdR처리적FOCUS세포주적HNF4αP1 mRNA상대표체량[(4.972±0.035)×10-6]고우공백대조조[(1.411±0.104)×10-6],차이유통계학의의(t=13.212,P＜0.01).경5-AZA-CdR연합TGF-β1처리적FOCUS세포주적HNF4αP1 mRNA상대표체량[(1.181±0.132)×10-6]저우단용5-AZA-CdR처리조[(4.972±0.035)×10-6],차이유통계학의의(t=13.873,P＜0.01).결론 간암조직중HNF4αP1표체수평하조.DNA갑기화가조공HNF4αP1재인간암세포주중적표체.HNF4αP1계동자구갑기화가억제TGF-β1대HNF4αP1표체적조절작용.
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.