Chen Yu-Qing, Zhao Cheng-Ling, Li Wei
Department of Respiration, First Affiliated Hospital of Bengbu Medical College, Bengbu, Anhui, PR China.
J Exp Clin Cancer Res. 2009 Feb 26;28(1):29. doi: 10.1186/1756-9966-28-29.
Survivin is a structurally and functionally unique member of the inhibitor of apoptosis protein (IAP) family. It plays an important role, not only in regulating mitosis but also in inhibiting apoptosis. The current literature contains few reports on the transcriptional regulation of survivin expression in lung cancer.
In this study, we investigated the effect of hypoxia-inducible factor-1alpha (HIF-1alpha) on the transcriptional activity of the survivin promoter in non-small cell lung cancer (NSCLC). Immunohistochemical staining was used to detect the expression of survivin and HIF-1alpha in the lung tissue of 120 patients with non-small cell lung cancer (NSCLC) and 40 patients with benign pulmonary disease. We also performed experiments with the lung adenocarcinoma cell line A549 cells, which were cultured under hypoxic conditions. The expression of survivin and HIF-1alpha was detected by real-time RT-PCR and Western blotting. In the survivin promoter the putative binding-site for HIF-1alpha, is -19 bpapproximately -16 bp upstream of TSS. We performed site-directed mutagenesis of this binding site, and used luciferase reporter plasmids to determine the relative activity of the survivin promoter in A549 cells. We also studied the effect of HIF-1alpha on the expression of survivin by dsRNA targeting of HIF-1alpha mRNA.
HIF-1alpha (58.33%) and survivin (81.60%) were both over-expressed in NSCLC and their expressions correlated with one another. They were also expressed in A549 cells under normal and hypoxic conditions, with a significant increase under hypoxic conditions. Site directed mutagenesis of the putative binding site for HIF-1alpha in the survivin promoter significantly decreased the activity of the survivin promoter in A549 cells. Inhibition of HIF-1alpha by RNAi decreased the expression of survivin in A549 cell lines.
Our results indicate that the binding of HIF-1alpha to the survivin promoter increases transcription of the survivin gene. Thus, HIF-1alpha is an important transcriptional regulator of survivin expression.
生存素是凋亡抑制蛋白（IAP）家族中结构和功能独特的成员。它不仅在调节有丝分裂中起重要作用，还在抑制细胞凋亡方面发挥作用。目前关于肺癌中生存素表达的转录调控的文献报道较少。
在本研究中，我们调查了缺氧诱导因子-1α（HIF-1α）对非小细胞肺癌（NSCLC）中生存素启动子转录活性的影响。采用免疫组织化学染色法检测120例非小细胞肺癌患者和40例良性肺疾病患者肺组织中生存素和HIF-1α的表达。我们还对肺腺癌细胞系A549细胞进行了实验，这些细胞在缺氧条件下培养。通过实时RT-PCR和蛋白质印迹法检测生存素和HIF-1α的表达。在生存素启动子中，HIF-1α的假定结合位点在转录起始位点（TSS）上游约-19 bp至-16 bp处。我们对该结合位点进行了定点诱变，并使用荧光素酶报告质粒来确定A549细胞中生存素启动子的相对活性。我们还通过靶向HIF-1α mRNA的dsRNA研究了HIF-1α对生存素表达的影响。
HIF-1α（58.33%）和生存素（81.60%）在NSCLC中均过度表达，且它们的表达相互关联。它们在正常和缺氧条件下的A549细胞中也有表达，在缺氧条件下显著增加。对生存素启动子中HIF-1α的假定结合位点进行定点诱变显著降低了A549细胞中生存素启动子的活性。RNAi抑制HIF-1α降低了A549细胞系中生存素的表达。
我们的结果表明，HIF-1α与生存素启动子的结合增加了生存素基因的转录。因此，HIF-1α是生存素表达的重要转录调节因子。
