缺氧诱导因子-2α与MMP-2启动子结合位点的确定

Determination of the binding site of hypoxia inducible factor-2α to the MMP-2 promoter

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250 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。

Purpose To identify the binding of hypoxia inducible factor-2α （HIF-2α） to the hypoxia-response element （HRE） of the matrixmetallo proteinases-2 （MMP-2） gene promoter region and clear the binding site. Methods Electrophoretic mobility shift assay （EMSA） was used to identify the binding of HIF-2α to HRE of the MMP-2 gene promoter region in vitro. At the same time, the chromatin immunoprccipitation assay （CHIP） was used to further determine the binding site. Results Successful prediction of two potential HIF-2α 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 a-bove 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-2α 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-2α binded to the FIRE in MMP-2 promoter region in vivo. Conclusion HIF-2α in MMP-2 promoter regionne promoter region in vitro and in vivo.