缺氧诱导因子-2α与MMP-2启动子结合位点的确定

摘要

Purpose To identify the binding of hypoxia in-ducible factor-2a (HIF-2 a) to the hypoxia-response element(HRE) of the matrixmetallo proteinases-2 (MMP-2) gene promoter region and clear the binding site. Methods Electro-phoretic mobility shift assay (EMSA) was used to identify the binding of HIF-2 a to HRE of the MMP-2 gene promoter region in vitro. At the same time, the chromatin immunoprecipitation assay (CHIP) was used to further determine the binding site. Results Successful prediction of two potential HIF-2a binding sites of MMP-2 the promoter region, which were-217～-204 and-1 029 ～-1 007, respectively. Probe test shows that the marked efficiency of sense chain and antisense chain was above 50％, and they could be used for EMSA-electrophoretic mobility shift assay. The results of EMSA showed that there was a binding site of HIF-2 a sense chain and antisense chain moter region int-217～-204. The results of chromatin immuno-precipitation showed that in the experimental group and control group an about 250 bp fragment in MMP-2 promoter containing HRE region was amplified, suggesting that the protein of HIF-2a binded to the HRE in MMP-2 promoter region in vivo. Conclusion HIF-2 a in MMP-2 promoter regionne promoter region in vitro and in vivo.%目的 探讨缺氧诱导因子-2α (hypoxia inducible factor-2α, HIF-2α)与基质金属蛋白酶-2 (matrixmetallo proteinases-2, MMP-2)基因中缺氧反应元件(hypoxia-response element, HRE)的体外结合,并确定该结合位点.方法 采用凝胶滞留电泳迁移率实验(electrophoretic mobility shift assay, EMSA)鉴定HIF-2α与MMP-2基因中HRE的体外结合;同时利用染色质免疫沉淀技术(chromatin immunoprecipitation assay, CHIP)进一步验证并确定该结合位点.结果 成功预测2条MMP-2启动子区潜在的HIF-2α结合位点,分别为-217 ～-204、-1 029～-1 007;探针标记效率检测结果显示,正义链和反义链标记效率均为50％以上,可用于EMSA实验;结果显示,MMP-2启动子区存在HIF-2α的结合位点,位于-217 ～-204.CHIP结果显示,实验组和对照组均扩增出约bp MMP-2启动子中含HRE区域的片段,提示HIF-2α蛋白与MMP-2启动子中的HRE区域存在活体内结合的关系.结论 HIF-2α与MMP-2基因中的HRE存在体内外结合.