RNA干扰沉默乏氧诱导因子1α对乏氧条件下食管鳞癌细胞化疗敏感性的影响

Impact of RNA interference targeting hypoxia-inducible factor-1α on chemosensitivity in esophageal squamous cell carcinoma cells under hypoxia

目的探讨 RNA 干扰技术沉默乏氧诱导因子(HIF)-1α对食管鳞癌 EC9706细胞乏氧条件下化疗敏感性的影响及其机制。方法应用化学乏氧法[氯化钴(CoCl_2)加入培养液的终浓度为75μmol/L]体外模拟肿瘤乏氧微环境。采用 MTS 比色法检测常氧培养组和乏氧培养组 EC9706细胞在顺铂/紫杉醇作用下细胞的抑制率,以及 RNA 干扰后乏氧培养细胞在顺铂/紫杉醇作用下细胞的抑制率。应用 Western 印迹方法检测 HIF-1α基因沉默后乏氧诱导 HIF-1α蛋白表达变化。用流式细胞仪检测 RNA 干扰前后乏氧条件下 EC9706细胞的细胞周期。结果针对 HIF-1α基因的 siRNA 干扰技术,有效抑制乏氧诱导的 HIF-1α蛋白表达。同一药物浓度常氧培养组细胞抑制率均明显高于乏氧培养组(均 P<0.05)。相同药物浓度及相同的乏氧条件下 RNA 干扰组抑制率均明显高于未转染组及转染 control siRNA 组(均 P<0.05)。相同的乏氧条件下转染 HIF-1αsiRNA 与未转染组/转染无关对照 siRNA 组相比,S 期显著增加(P<0.05)、(G1期细胞减少(P<0.05)。结论乏氧条件下 HIF-1α诱导 EC9706细胞周期停止,可能是乏氧条件下细胞耐药的机制之一。RNA 干扰可逆转食管痛细胞EC9706乏氧环境中化疗耐药。

Objective To investigate the impact of RNA interference （RNAi） targeting hypoxiainducible factor 1 alpha （HIF-1 α） on chemosensitivity of esophageal squamous cell carcinoma cells under hypoxia. Methods Human esophageal squamous cell carcinoma cells of the line EC9706 were cultured and divided into 3 groups： untransfected group, added with cobalt chloride （CoCl2）, a chemical hypoxia inducer, for 8 h so as to establish a hypoxia model; control siRNA transfected group, transfected with control siRNA, and 30 h after the transfection exposed to CoCl2 for 8 h; and HIF-1 α siRNA-transfected group, transfected with HIF-1 α siRNA, and 30 h later exposed to CoCl2 for 8 h. Western blotting was used to detect the protein expression of HIF-1α. Another EC9706 were cultured and divided into 3 groups to be treated as mentioned above, and then exposed to cisplantin or platixal under normoxic or hypoxic condition. 24 hours later 3-（4, 5-carboxymethoxyphenyl ）-2-（4-sulfophenyl）-2H-tetrazolium （MTS） colorimetric assay was used to detect the inhibition rates of the cells. Further another EC9706 cells were cultured and then divided into 5 groups： cultured under normoxic condition, cultured under hypoxic condition for 8 h, transfected with control siRNA for 30 h and then under hypoxic condition for 8 h, transfected with HIF-1 α siRNA for 30 h and then under hypoxic condition for 8 h. The cell cycle was measured by flow cytometry. Results The HIF-1α protein expression of the HIF-1α siRNA group was significantly lower than those of the untransfected and control siRNA transfected groups. The inhibition rates of the EC9706 cells of the groups treated by cisplatin of different concentrations under normoxic condition were all significantly higher than the corresponding levels under hypoxic condition （all P 〈 0.01 ）. The inhibition rates of the EC9706 cells of the groups treated by platixal of different concentrations under normoxic condition were all significantly higher than the corresponding levels under hypoxic condition （ all P 〈 0.05 ） Under hypoxic condition, the inhibition rates of the HIF-1 α siRNA transfected EC9706 cells treated by cisplatin and platixal of different concentrations were all significantly higher than those of the control siRNA transfected and untransfected EC9706 cells （ all P 〈 0. 05 ）. Flow cytometry showed that under hypoxic condition the proportion of cells in Gl-phase of the EC9706 cells was significantly higher, and the proportion of S-phase cells was significantly lower than those of the normoxic group （ both P 〈 0.05 ）, and under the same hypoxic condition the proportion of the EC9706 cells in Gl-phase was significantly lower, and the proportion the EC9706 cells in S-phase was significantly higher than those of the normoxic group （ all P 〈 0. 05 ）. Conclusion The cell cycle arrest induced by HIF-1α may be the mechanism of the resistance to anticancer drugs of the esophageal squamous cell carcinoma cells under hypoxic condition. Blocking HIF-1α lpha in esophageal squamous cell carcinoma cells may reverse the muhidrug resistance of the tumor cells, so it may offer an avenue for gene therapy.