CAJ | 학술논문

目的探讨Twist在肝细胞癌中的表达及其与缺氧诱导因子(HIF)-1α的关系,观察Twist和HIF-1α基因干扰对肝细胞癌HepG2细胞增殖和凋亡的影响.方法免疫组织化学法检测106例肝细胞癌组织中转录因子Twist和HIF-1 α的表达;利用LipofectamineTM 2000转染Twist和HIF-1α小分子干扰RNA(siRNA)至HepG2细胞.实验分5组:Twist干扰组、HIF-1α干扰组、双重干扰组、阴性对照组(转染negative-siRNA)和空白对照组(转染脂质体);流式细胞仪检测细胞凋亡;噻唑蓝(MTT)比色法检测细胞体外增殖活力;染色质免疫共沉淀法(ChIP)检测Twist和HIF-1α基因间的直接作用关系.结果癌组织Twist的阳性表达率为66.98％,明显高于癌旁组织的17.92％(P＜0.05),Twist和HIF-1α的表达呈正相关(P＜0.05).与空白对照组(3.98％)和阴性对照组(4.26％)比较,Twist干扰组、HIF-1 α干扰组和双重干扰组细胞凋亡率分别为41.62％、43.84％和45.12％,干扰组细胞凋亡率均显著增加(P＜0.01),且干扰组之间差异无统计学意义(P＞0.05).Twist干扰组、HIF-1α干扰组和双重干扰组细胞生长抑制率分别为30.2％、34.0％和35.8％,各干扰组间差异无统计学意义(P＞0.05).ChIP法证实在HepG2中Twist与HIF-1α启动子有直接作用.结论 Twist与HIF-1α基因表达呈正相关,表达下调后能诱导肝细胞癌HepG2细胞凋亡,并抑制其生长,双重干扰无明显协同作用,并且两基因间具有直接作用.
목적 탐토Twist재간세포암중적표체급기여결양유도인자(HIF)-1α적관계,관찰Twist화HIF-1α기인간우대간세포암HepG2세포증식화조망적영향.방법 면역조직화학법검측106례간세포암조직중전록인자Twist화HIF-1 α적표체;이용LipofectamineTM 2000전염Twist화HIF-1α소분자간우RNA(siRNA)지HepG2세포.실험분5조:Twist간우조、HIF-1α간우조、쌍중간우조、음성대조조(전염negative-siRNA)화공백대조조(전염지질체);류식세포의검측세포조망;새서람(MTT)비색법검측세포체외증식활력;염색질면역공침정법(ChIP)검측Twist화HIF-1α기인간적직접작용관계.결과 암조직Twist적양성표체솔위66.98％,명현고우암방조직적17.92％(P＜0.05),Twist화HIF-1α적표체정정상관(P＜0.05).여공백대조조(3.98％)화음성대조조(4.26％)비교,Twist간우조、HIF-1 α간우조화쌍중간우조세포조망솔분별위41.62％、43.84％화45.12％,간우조세포조망솔균현저증가(P＜0.01),차간우조지간차이무통계학의의(P＞0.05).Twist간우조、HIF-1α간우조화쌍중간우조세포생장억제솔분별위30.2％、34.0％화35.8％,각간우조간차이무통계학의의(P＞0.05).ChIP법증실재HepG2중Twist여HIF-1α계동자유직접작용.결론 Twist여HIF-1α기인표체정정상관,표체하조후능유도간세포암HepG2세포조망,병억제기생장,쌍중간우무명현협동작용,병차량기인간구유직접작용.
Objective To investigate the expression of Twist in hepatocellular carcinoma (HCC),the relationship between Twist and hypoxia inducible factor (HIF)-lα,and the effect of silencing Twist and HIF-1 α genes on the proliferation and apoptosis of HCC HepG2 cells by RNA interference.Methods The expression of Twist and HIF-1α was detected in 106 cases of HCC by immunohistochemical staining method.Twist and HIF-1α-small interfering RNA (siRNA) were transfected into HepG2 cells mediated by LipofectamineTM 2000.HepG2 cells were divided into five groups:Twist interference group,HIF-1 α interference group,double interference group,negative control group (transfected with negative-siRNA) and blank control group (transfected with Lipofectamine).Cell apoptosis was examined by flow cytometry and cell growth was measured by methyl-thiazol-tetrazolium (MTT) assay.The direct interactions between Twist and HIF-1α were uncovered by chromatin immunoprecipitation assay (ChIP).Results Twist positive expression rate was high (66.98％) in HCC tissue but low in the corresponding peritumoral tissue (17.92％,P＜0.05).There was a positive relationship between the expressions of Twist and HIF-1α(P ＜ 0.05).The apoptosis rate in blank control group,negative control group,Twist interference group,HIF-1o interference group and double interference group was 3.98％,4.26％,41.62％,43.84％,and 45.12％,respectively.The apoptosis rate in interference groups was significantly higher than that in control groups (P ＜ 0.01),while there was no significant difference among three imerference groups (P ＞0.05).The cell growth inhibitory rate in Twist interference group,HIF-1α interference group and double interference group was 30.2％,34.0％ and 35.8％,respectively (P ＞0.05).There were the direct interactions between Twist and HIF-1α promoter in HepG2 by ChIP.Conclusion There was a positive relationship between the expression of Twist and HIF-1α.Down-regulation of Twist and HIF-1α could induce apoptosis of HepG2 cells and inhibit cell growth.The double knockdown of Twist and HIF-1 α does not show a synergistic effect,and there were direct interactions between Twist and HIF-1α.