低氧诱导因子-1αRNAi表达质粒对卵巢癌细胞影响的实验研究

聂春莲; 高国兰

doi:10.3969/j.issn.1000-8179.2011.09.002

摘要: 目的：探讨人HIF-1α短发夹（sh） RNA表达质粒对卵巢癌SKOV3细胞HIF-1α基因的表达及对紫杉醇敏感性的影响。方法：构建HIF-1α （sh） RNA表达质粒，脂质体法转染， RT-PCR和Western blot检测癌细胞HIF-1α mRNA和蛋白表达水平，MTT法检测癌细胞对紫杉醇的敏感性。结果：转染质粒48 h后， H质粒转染组SKOV3细胞紫杉醇作用24 h的IC50值明显降低（P<0.05），同时P-糖蛋白表达水平显著降低，差异有统计学意义（F=7.24， P=0.01）。结论：针对HIF-1α的RNAi表达质粒可下调低氧培养的卵巢癌细胞SKOV3 HIF-1α基因mRNA和蛋白及P-gp的表达，从而提高癌细胞对紫杉醇的敏感性。

Abstract: Effect of Hypoxia Inducible Factor-1 α RNAi Expressing Plasmid on Ovarian Cancer SKOV3 CellsChunlian NIE1, Guolan GAO2Correspondence to: Guolan GAO, E-mail: guolan_gao@yahoo.com.cn1Department of Obstetrics and Gynecology, Chongqing Hospital for Maternal and Child Health Care, Chongqing 400013, China2Department of Obstetrics and Gynecology, The First Affiliated Hospital of Nanchang University, Nanchang 330006, ChinaGrant support: This study was supported by National Natural Science Foundation of China (No.30660192) and Jiangxi PostgraduateStudent Innovation Funds (No. YC07A018)Abstracts Objective: To investigate the effect of HIF-1α short-hairpin RNA (shRNA) expressing plasmid on ovarian cancer invitro. Methods: Oligos for hairpin RNA expression which targeted HIF-1α gene were designed and selected based on the well-knownprinciple, using online softwares. Annealed oligos were inserted into the pGPU6/GFP/Neo plasmid to construct recombinant plasmids.The plasmids were transfected into SKOV3 cells using lipofectmine. The expression of HIF-1α mRNA and protein was assayed byRT-PCR and Western blot respectively. Chemotherapy drug sensitivity was determined by methylthiazolyltetrazolium ( MTT ) assay.Results: At 48 hours after plasmid transfection, compared with that of Lipo group and NC group, IC50 was significantly lower inSKOV3 cells of the H plasmid transfection group undergoing a 24-hour Paclitaxel medication ( P < 0.05 ). The expression of P-gp wasobviously lower in SKOV3 cells of the H plasmid group than in the Lipo and NC group, with significant difference ( F = 7.24, P =0.01 ). Conclusion: RNAi expressing plasmid directly against HIF-1α can effectively down-regulate the expression of HIF-1α mRNAand protein in SKOV3 cells. After selectively silencing HIF-α, hypoxia-induced expression of its target genes such as P-glycoproteincan be markedly attenuated. HIF-1α down-regulation could increase the drug sensitivity of SKOV3 cells to Paclitaxel.Keywords Hypoxia; RNA interference; Hypoxia inducible factor-1 α; Ovarian neoplasms; P-glycoprotein