RNA干扰HIF-1α影响宫颈癌HeLa细胞上皮细胞间质变标记蛋白的研究

Effect of HIF-1α RNA Interference on Expression of EMT-associated Proteins in Human Cervical Carcinoma HeLa Cells

目的 通过RNA干扰技术沉默人宫颈癌HeLa细胞缺氧诱导因子-1α（hypoxia inducible factor-1α,HIF-1α）的表达,在细胞水平研究HIF-1α对宫颈癌HeLa细胞上皮细胞-间质变标记蛋白（E-cadherin,N-cadherin,vimentin）表达的影响.方法 将人宫颈癌HeLa细胞株（H0细胞）、转染pGenesil-1空白质粒的HeLa细胞株（H1细胞）及转染HIF-1α-shRNA质粒的HeLa细胞株（H2细胞）分别行常氧及缺氧（150 μmol/L CoCl2培养12 h）培养,应用Western印迹、免疫细胞化学法检测每组HeLa细胞中HIF-1α、上皮标记蛋白E-cadherin、间质标记蛋白vimentin、N-cadherin的表达情况.结果 Western印迹分析各缺氧组HIF-1α、E-cadherin、vimentin及N-cadherin的平均光密度值,免疫细胞化学显示H0细胞、H1细胞缺氧时HIF-1α、N-cadherin、vimentin蛋白高表达,而E-cadherin低表达,H2细胞在乏氧时HIF-1α、N-cadherin、vimentin蛋白表达显著减弱,而E-cadherin高表达.结论 通过shRNA干扰抑制人宫颈癌HeLa细胞HIF-1α,可一定程度上抑制乏氧环境中宫颈癌HeLa细胞上皮细胞-间质变.

Objective To investigate the effect of RNA interference （RNAi） targeting hypoxia inducible factor-1α（ HIF-1α ） on epithelial-meseuchymal transition （EMT） -associated proteins in human cervical carcinoma HeLa Cells. Methods Human cervical carcinoma HeLa cell line （H0 cells）, pGenesil-1 transfected HeLa cell line （HI ceils） and HIF-1α-shRNA transfected HeLa cell line （H2 cells） were cuhured under hypoxia （150μmol/LCoCl2） and normoxia for 12 h respectively. Expression of HIF-1α, E-cadherin, N-cadherin and Vimentin in each group was detected by western hiotting and immunohistochemical staining. Results Western blotting showed that under hypoxic condition the expression density of HIF-1α, E-cadherin, N-cadherin and vimentin were 1.182±0.251, 0.409 ±0. 112, 3. 187 ±0.523 and 1.638 ±0.417 in HO cells, 1.364± 0.279, 0.441±0.126, 3.276±0.601 and 1.437±0.279 in H1 cells, and 0.248 ±0.053, 0. 833 ± 0. 257, 0. 992 ± 0. 270 and 1. 004± 0. 236 in H2 cells respectively. Immunohistochemical staining showed that, under hypoxic condition, expression of HIF-1α, N-cadherin and Vimentin was increased in HO and H1 cells but decreased in H2 cells, whereas E-cadherin expression was decreased in 1α and H1 cells but increased in H2 cells. Conclusion RNA interference of hypoxia inducible factor-1α by may suppress EMT of human cervical carcinoma HeLa cells under hypoxic condition.