摘要
目的恶性肿瘤在生长过程中会出现肿瘤内部缺氧微环境,缺氧诱导因子1α(hypoxia-inducible factor 1α,HIF-1α)和缺氧诱导因子2α(hypoxia-inducible factor 2α,HIF-2α)等因子能够适应细胞内缺氧微环境,并对多种基因进行调控,促进肿瘤细胞增殖、迁移侵袭以及放化疗抵抗等。本研究旨在探讨siRNA沉默HIF-2α后对人肝癌细胞株HepG2增殖、侵袭和细胞周期素D1(Cyclin D1)表达的影响。方法 200μmol/L氯化钴(CoCl2)诱导细胞模拟缺氧,作为低氧组,HIF-2αsiRNA转染缺氧环境下HepG2细胞,分为低氧+HIF-2αsiRNA组、低氧+Control siRNA组,Real-time PCR、蛋白质印迹法检测转染48h后各组细胞中HIF-2α、Cyclin D1的mRNA和蛋白表达,MTT法检测HepG2细胞增殖,流式细胞术观察细胞周期分布,侵袭试验观察细胞侵袭能力。结果转染特异性HIF-2αsiRNA于HepG2细胞后,低氧+HIF-2αsiRNA组HIF-2α和Cyclin D1mRNA相对表达量分别为0.557±0.082和0.352±0.049,显著低于低氧组1.127±0.069和0.740±0.068,t值分别为13.092和11.391,均P<0.001;HIF-2α和Cyclin D1蛋白相对表达量分别为0.018±0.005和0.338±0.054,显著低于低氧组0.077±0.010和0.613±0.047,t值分别为12.728和9.411,均P<0.001;低氧+HIF-2αsiRNA组HepG2细胞在24、48、72、96和120h的A值分别为0.335±0.045、0.485±0.080、0.631±0.063、0.772±0.055和0.651±0.080,显著低于低氧组0.512±0.061、0.726±0.054、0.836±0.072、0.867±0.077和0.785±0.063,均P<0.001;低氧+HIF-2αsiRNA组穿膜细胞数(37.83±4.920)显著低于低氧组(86.17±7.030),t=13.806,P<0.001;合成期(S期)与合成后期(G2/M期)细胞比率分别为(21.168±2.007)%和(18.187±5.500)%,均低于低氧组(26.048±3.558)%和(27.843±3.193)%,t值分别为2.926和3.719,P值分别为0.015和0.004,G0/G1期细胞比率为(60.645±4.199)%,显著高于低氧组(46.108±4.556)%,t=5.748,P<0.001。结论HIF-2α基因表达水平下降可抑制HepG2细胞增殖侵袭和改变细胞周期分布,可能与下调Cyclin D1的表
OBJECTIVE The tumor hypoxia microenvironment can be induced within the growth process of malig-nant tumors. Some factors such as hypoxia inducible factor 1 alpha（HIF-1α） and hypoxia inducible factor 2 alpha（HIF-2α） can adapt to cell hypoxia microenvironment and regulate a variety of genes. Meanwhile they can also promote proliferation, migration,invasion and resistance to chemotherapy of tumor cells. We aimed to discuss the siRNA silencing of HIF-2α on the proliferation and invasion of human hepatoma cell line HepG2 as well as the expression of Cyclin D1. METHODS Cells were treated by 200 -mol/L cobalt chloride （COCl2） to simulate hypoxia condition as hypoxic control group, HIF-2α siRNA transfection of HepG2 cells under hypoxia environment,divided into hypoxia plus HIF-2α siRNA group, and hypoxia plus control siRNA group. Real-time PCR,Western blot method were used to detect the transfection of ceils in each group after 48h of HIF-2α,Cyclin D1 mRNA and protein expression; MTT method was used to detect the proliferation of HepG2 cells;distribution of cell cycle was detected by flow cytometry;cell invasion was observed by inva- sive test. RESULTS After the specific HIF-2α siRNA was transfected to the HepG2 cells,the expression of HIF-2α and Cyclin D1 mRNA in the bypoxic plus HIF-2α siRNA group were 0. 557±0. 082 and 0. 352±0. 049,which were significantly lower than that of hypoxia group （1. 127±0.069, 0.740 ±0. 068, t = 13. 092, t=11. 391, respectively, all P 〈 0. 001）;expression of HIF-2α and Cyclin D1 protein in the hypoxic plus HIF2α siRNA group were 0. 018±0. 005 and 0. 338±0. 054,which were significantly lower than that of hypoxia group（0. 077±0. 010,0. 613±0. 047,t= 12. 728,t= 9. 411 ,respectively,all P〈0. 001）. A value of HepG2 cells in the hypoxic plus HIF-2α siRNA group at 24,48,72,96, 120 h were 0. 335!0. 045,0. 485±0. 080,0. 631±0. 063,0. 772±0. 055,0. 651±0. 080,which were lower than that of hypoxia group 0. 512±0. 061,0. 726±0. 054,0. 836±0. 072,0. 867
作者
李伟伟
耿晓松
孙建伟
张彩凤
申保生
宋新文
LI Wei-wei;GENG Xiao-song;SUN Jian-wei;ZHANG Cai- feng;SHEN Bao-sheng;SONG Xin-wen(First Affiliated Hospital, Xinxiang Medical University ,Weihui 453100 ,P. R. China;School of Nursing, Xinxiang Medical University, Xinxiang 453003, P. R. China)
出处
《中华肿瘤防治杂志》
CAS
北大核心
2018年第7期476-480,484,共6页
Chinese Journal of Cancer Prevention and Treatment
关键词
肝癌
缺氧诱导因子2α
RNA干扰
细胞增殖
细胞周期素D1
human hepatocellular carcinoma
hypoxia induced factors 2a
RNA interferenee
cell proliferation
Cyclin D1
