摘要
目的：分析肝癌组织缺氧诱导因子-1α（HIF-1α）表达特征及沉默HIF-1α对癌细胞增殖的影响.方法：以免疫组化法分析自身配对的肝癌及癌周组织中HIF-1α表达.HepG2细胞转染靶向HIF-1α的miRNA干扰质粒,RT-PCR、Western blot检测HIF-1α基因及蛋白水平变化;酶联免疫吸附法检测培养液血管内皮生长因子（VEGF）、血管生成素（ANG）-2表达;阿霉素预处理后,以Annexin V-FLUOS/PI双染法分析HepG2细胞凋亡率,并检测细胞周期改变.结果：肝癌及周围组织HIF-1α呈棕黄色颗粒状表达,定位于胞浆和胞核,其特征是癌旁组织全数表达高于癌灶组织（80.0%,χ2=22.35,P＜0.001）,癌灶组织HIF-1α表达与肿瘤大小相关.HepG2细胞转染靶向HIF-1α miRNA干扰质粒后72 h,HIF-1α在转录水平下降87%,蛋白水平下降56%;培养液中VEGF和ANG-2表达水平分别减少54%和36%;HepG2细胞凋亡率为（22.46±0.61）%,G1期和S期比率分别为（61.49±1.12）%和（22.40±0.58）%;联合阿霉素后HepG2细胞凋亡率为（36.99±0.88）%,G1期和S期比率分别为（65.68±0.91）%和（19.47±1.34）%.结论：肝癌组织HIF-1α过表达与肝癌发展相关,沉默HIF-1α可有效调控下游血管生成相关基因表达,抑制癌细胞增殖,改善化疗药物敏感性.
Objective： To analyze the expression features of the hypoxia-inducible factor-1α （ HIF-1α ） in hepatocellular carcinoma （ HCC ） tissues and the effects of HIF-1α silencing on HepG2 cells. Methods： HIF-1α expression was analyzed in the self-control HCC specimens via immunohistochemistry. At 72 h after transfection with the HIF-1α miRNA plasmid, the levels of HIF-1 ct mRNA and protein in the HepG2 cells were determined using real-time polymerase chain reaction （ PCR ） and Western blot analysis. The vascular endothelial growth factor （VEGF） and angiopoietin-2 （ ANG-2 ） expressions in the supernate were determined using ELISA. Moreover, the alterations in the cell cycles and apoptosis in the HepG2 cells with miRNA and doxorubicin were measured using a flow cytometer. Results： The positive HIF-1α was brown and granular in the cytoplasm or nucleus. A significant difference was found between HCC （ 80% ） and its surrounding tissues （ 100%, x2 = 22.35, P 〈 0.001 ）. The features of HIF-1α were correlated with tumor size. At 72 h after transfection, the expressions of HIF-1α mRNA and protein in HepG2 were down-regulated by 87% and 56%, respectively. VEGF and ANG-2 were also reduced by 54% and 34%, respectively. After a combined doxorubicin and HIF-1α silencing, the apop- totic ratio increased from 22.46% - 0.61% to 36.99% -0.88%. The proportions of the GI and S phases were up-regulated to 65.68% 0.91% and 19.47%-1.34%, respectively. Conclusion： The abnormal expression of HIF-1α is associated with the occurrence and development of HCC. In addition, HIF-1α silencing can effectively inhibit HepG2 proliferation.
出处
《中国肿瘤临床》
CAS
CSCD
北大核心
2011年第14期830-834,共5页
Chinese Journal of Clinical Oncology
基金
江苏省自然科学基金(编号:BK2008187)
南通市社会发展项目(编号:S2009027)资助~~
关键词
肝癌
缺氧诱导因子-1Α
表达
RNA干扰
基因沉默
Hepatocellular carcinoma
Hypoxia-inducible factor-1α
Expression
RNA interference
Gene silencing
