Xia Li-Min, Tian De-An, Zhang Qiong, Yan Wei, Zhu Qian, Luo Min, Zhou Zhen-Zhen, Tang Ying, Zhang Quan-Le, Wang Wei
Division of Gastroenterology, Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
Zhonghua Gan Zang Bing Za Zhi. 2009 Mar;17(3):207-12.
To investigate role of hypoxia inducible factor 1 (HIF-1) in the transcriptional activation of heat shock protein 70-2 (HSP70-2) in hepatocellular carcinoma (HCC) cells under hypoxic conditions.
HCC cells were exposed to reduced oxygen atmosphere (1% O2), or treated with YC-1 or HIF-1 alpha siRNA, the expression of HIF-1 alpha and HSP70-2 were detected by Western blot analysis. Serial deletions of the HSPA2 promoter were cloned in the reporter pGL3-Basic plasmid. These reporter plasmids were co-transfected with HIF-1 alpha siRNA, and the promoter activities were detected with the dual luciferase assay.
Western blot analysis showed that both HIF-1 alpha and HSP70-2 proteins were strongly increased after HCC cells were exposed to hypoxic conditions (1% O2) for 6 h, and the expression level of HSP70-2 was increased in a time-dependent manner. Treatment of HepG2 cells with YC-1 or HIF-1 alpha siRNA significantly inhibited the expression of HIF-1 alpha and HSP70-2. In silico analysis of the HSP70-2 promoter using the Gene2 Promoter software revealed the presence of two putative hypoxic response element (HRE) consensus at -446bp (HRE1) and -238bp (HRE2). Depletion of promoter sequence between -653 and -385 led to a dramatic reduction of promoter activity, whereas further deletion to position -201 did not reduce the activity further. These data suggested that HRE1 plays an important role in hypoxia-induced activation of the HSPA2 promoter. Site-directed mutagenesis further confirmed these results. Mutation of HRE1 but not of HRE2 abrogated the sensitivity of the HSP70-2 promoter to hypoxia.
HSP70-2 expression is up-regulated in response to hypoxia and a HIF-1 binding site (HRE1) in the HSP70-2 promoter is involved in this response.
研究缺氧诱导因子1（HIF-1）在缺氧条件下对肝癌（HCC）细胞中热休克蛋白70-2（HSP70-2）转录激活的作用。
将HCC细胞置于低氧环境（1% O₂）中，或用YC-1或HIF-1α小干扰RNA（siRNA）处理，通过蛋白质免疫印迹分析检测HIF-1α和HSP70-2的表达。将HSPA2启动子的系列缺失片段克隆到报告载体pGL3-Basic质粒中。这些报告质粒与HIF-1α siRNA共转染，并用双荧光素酶测定法检测启动子活性。
蛋白质免疫印迹分析显示，HCC细胞在缺氧条件（1% O₂）下暴露6小时后，HIF-1α和HSP70-2蛋白均显著增加，且HSP70-2的表达水平呈时间依赖性增加。用YC-1或HIF-1α siRNA处理HepG2细胞可显著抑制HIF-1α和HSP70-2的表达。使用Gene2 Promoter软件对HSP70-2启动子进行电子分析，发现在-446bp（HRE1）和-238bp（HRE2）处存在两个假定的缺氧反应元件（HRE）共有序列。-653至-385之间的启动子序列缺失导致启动子活性显著降低，而进一步缺失至-201位置并未进一步降低活性。这些数据表明，HRE1在缺氧诱导的HSPA2启动子激活中起重要作用。定点诱变进一步证实了这些结果。HRE1的突变而非HRE2的突变消除了HSP70-2启动子对缺氧的敏感性。
HSP70-2的表达在缺氧时上调，且HSP70-2启动子中的一个HIF-1结合位点（HRE1）参与了这一反应。
