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的表达有关。
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±0. 077,0. 785±0. 063,the differences were statistically significant（all P〈0. 001）. Number of transmembrance cells in hypoxia plus HIF-2α siRNA group（37.83±4. 920） was significantly lower than that in bypoxia group（86.17 ± 7. 030）, the differences was statistically significant（t= 13. 806, P〈0. 001）. Cell ratio of S stage,G2/M stage were（21. 168±2. 007） %, （18. 187±5. 500）%,which were significantly lower than that in hypoxia group（26. 048±3. 558）%, （27. 843±3. 193）%, the differences were statistically significant （t = 2. 926, P= 0. 015 ;t= 3. 719, P=0. 004, respectively）. Cell ratio of Go/G1 stage was （ 60. 645 ± 4. 199）% in hypoxia plus HIF-2α siRNA group,which was significantly higher than that in hypoxia group （46. 108±4. 556）%, the differences was statistically significant（t=5. 748,P〈0. 001）. CONCLUSION HIF2α gene expression levels can inhibit HepG2 cell proliferation,invasion and cell cycle distribution,and may be associated with reduced expression of Cyclin D1.
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
