Background and purpose: Survivin gene is a unique member of the inhibitor of apoptosis protein (LAP) family. It plays an important role, not only in regulating mitosis but also in inhibiting apoptosis. It is highly expressed in almost all types of human tumors and fetal tissues but rarely detectable in normal adult tissues. High levels of survivin expression have been associated with tumor progression, resistance to radiation and drug treatments and poor survival rates in cancer patients. The current literature contains few reports on the transcriptional regulation of survivin expression in lung cancer. Previous studies have found that there are also 2 putative binding sites for hypoxia-inducible factor- la(HIF- la) in the core promoter region of survivin gene. Survivin promoter-luciferase reporter vectors Pgl3-SVP230-luc have been constructed early. The purpose of this study was to investigate the mechanism of (HIF-la)on transcriptional regulation of survivin in A549 cells by hypoxia. Methods: (l)Double labeling immunofluorescence method was used to detect co-expression of survivin/HIF-lα protein; (2)RT-polymerase chain reaction (RT-PCR) and Western blot was used to examine the level of survivin Mrna and protein in A549 cells transfected by HIF-lα expression plasmid and HIF-lα siRNA; (3)Luciferase activity was detected in A549 cells following cotransfection with Pgl3-SVP230-luc as well as HIF-la expression plasmid or HIF-lα siRNA to value the transcriptional activity of survivin. (4)Electrophoretic mobility shift assay (EMS A) was performed to test the nuclear extract of the A549 cells binding to the r-32P labeled probes containing survivin promoter squences. Results: (l)Survivin/HIF-lα proteins co-expressed in A549 cell; (2)Compared with control groups, the level of survivin Mrna and protein is markedly increased in A549 cells transfected with HIF-lα expression plasmid, but decreased in the HIF-lα siRNA group(P<0.01); (3)The relative activity of Pgl3-SVP230-luc in A549 cells transfected with HIF-lα expression plasmid was 78.84, which was significantly higher than that of both the pcDNA3.0 and the negative control group in the A549 cells (P<0.01), but was significantly lower in the HIF-la siRNA group (P<0.01); (4)DNA-neucleoprotein bands were observed when A549 nuclear extracts incubating with the r-32P labeled -18 bp probe (nucleotides -26 to -9) of survivin core promoter in EMSA assay. The specific bands were competed away by the cold 18 bp probe, but not the mutated cold probe in competition assay. Binding bands exhibited in Super-shift reaction. (5)The proliferation of A549 cells was inhibited after transfection with HIF-la siRNA. Cell apoptosis was significantly increased in HIF-la siRNA group compared to negative control group (P<0.01). Conclusion: The binding of HIF-la to the survivin core promoter (nucleotides-19 to -16 bp in its 5' flanking region) increases transcription and expression of the survivin gene. These observations have significant implications for understanding the part molecular mechanism of survivin transcriptional regulation in lung cancer.%背景与目的:survivin是一种重要的抗凋亡基因,在肿瘤组织中特异性高表达,并参与调控细胞周期和细胞凋亡,其高表达与肿瘤进展、药物抵抗以及预后关系密切.但survivin在肺癌中高表达的转录调控机制研究甚少.课题组前期研究成功构建含有survivin核心启动子的荧光报告载体pGL3-SVP230-1uc,并发现核心启动子区存在缺氧诱导因子-1α(hypoxia-inducible factor-1 α,HIF-1 α)的潜在位点.本研究拟通过免疫荧光双标记法、电泳迁移率实验(EMSA)、共转染等方法探讨缺氧条件下人肺腺癌细胞株A549中HIF-1 α调控survivin转录的确切机制.方法:(1)运用免疫荧光双标记检测缺氧A549细胞中HIF-1 α与survivin的表达；(2)HIF-1α表达质粒和HIF-1α siRNA分别转染A549细胞,30 h后RT-PCR、Western blot和免疫荧光双标法检测survivin mRNA和蛋白表达；(3)pGL3-SVP230-luc(含有survivin核心启动子的荧光报告载体)与HIF-1 α表达质粒和HIF-1α siRNA分别共转染A549细胞,测定萤光素酶的表达活性；(4)EMSA分析HIF-1 α与survivin核心启动子结合情况；(5)流式细胞术和MTT法检测细胞凋亡和增殖.结果:(1)HIF-1 α与survivin共表达于A549细胞中；(2)转染HIF-1α表达质粒组survivin mRNA和蛋白表达水平显著上调,转染HIF-1 α siRNA组survivin mRNA和蛋白表达显著降低(P＜0.01)；(3)HIF-1 α表达质粒组pGL3-SVP230-luc相对活性分别为78.84,显著高于空质粒组及对照组(P＜0.01),HIF-1 α siRNA组pGL3-SVP230-luc相对活性分别为28.84,显著低于空质粒组及对照组(P均＜0.01);(4)HIF-1α与survivin核心启动子区转录起始点上游-19～-16 bp序列结合转录激活survivin(5)流式细胞术和MTT检测结果显示,HIF-1 α siRNA组细胞凋亡显著增加,细胞抑制率明显升高,与对照组相比差异具有统计学意义(P均＜0.01).结论:缺氧状态下A549细胞中survivin与HIF-1 α存在共表达；HIF-1 α可以通过survivin核心启动子区的结合位点激活survivin转录和表达;靶向HIF-1 α可以诱导细胞凋亡、抑制细胞增殖,并为survivin的靶向治疗研究提供了进一步的实验基础.
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