层黏连蛋白受体参与缺氧诱导胃癌多药耐药的机制研究

The role of 67LR in hypoxia-induced MDR in gastric cancer

目的:研究缺氧对胃癌细胞对于化疗药物敏感性的影响以及层黏连蛋白受体在缺氧诱导的胃癌MDR中的作用和分子机制。方法:MTT比色法、Annexin V/PI染色法和阿霉素的蓄积和潴留实验检测胃癌细胞在缺氧和常氧状态下对化疗药物敏感性的差异;Western blot和半定量RT-PCR检测缺氧条件下胃癌细胞中67Kda层黏连蛋白受体(67Kda laminin receptor,67LR)的表达;Western blot、半定量RT-PCR方法检测缺氧条件下胃癌细胞系SGC7901中67LR的表达和活性;利用siRNA干涉67LR的表达,MTT比色法、AnnexinV/PI染色法检测缺氧条件下调下胃癌细胞系67LR的表达对化疗药物敏感性的影响。结果:缺氧能够显著降低胃癌细胞对化疗药物的敏感性,以及化疗药物诱导的凋亡和药物在细胞内的潴留和蓄积;缺氧能够显著上调67LR的表达和转录活性;67LR siRNA能够抑制LR的表达;抑制67LR的表达能够显著逆转缺氧诱导的胃癌的MDR表型。结论:缺氧能够增加胃癌细胞对于化疗药物的抵抗,通过上调67LR表达,加剧了缺氧诱导的胃癌多药耐药表型,抑制67LR的表达能够逆转缺氧诱导的胃癌多药耐药的发生。

Objective：To study hypoxia-induced chemotherapeutic drug sensitivity,and the mechanism of hypoxia-induced drug resistance in gastric cancer cells,characterize the role and illuminate the molecular mechanisms of 67LR contributing to hypoxia-induced MDR.Methods：MTT assay,Annexin V/PI staining and Adriamycin accumulation and retention were used to determine the sensitivity of cells to chemotherapeutic drugs under normoxic and hypoxic condition.By semiquantitative RT-PCR and Western blot,the expression of 67LR was measured in the cells exposed to hypoxic condition at indicated times.Semiquantitative RT-PCR,Western blot assay and dual luciferase reporter assay were used to study the induction of 67LR by hypoxia.Under hypoxic condition,MTT assay and Annexin V/PI staining were used to determine the drug sensitivity in gastric cancer cells introduced 67LR siRNA vector.Results：Under hypoxic condition,gastric cancer cells revealed an increased drug resistance toward different chemotherapeutic drugs,a decrease apoptosis index induced by chemotherapeutics and a decrease accumulation and retention of ADR.Semiquantitative RT-PCR and Western blot results revealed that hypoxia increased 67LR mRNA and protein expression.Hypoxia could induce 67LR mRNA and protein changes and transcriptional activity.Cells blocKing MGr-1Ag expression by stable transfection of 67LR siRNA vector inhibited hypoxia-induced drug resistance,abolished hypoxia-prevented VCR-induced apoptosis.Conclusion：Hypoxia induced 67LR upregulation and subsequently promoted multidrug resistance in gastric cancer cells.