Effect of hypoxia-inducible factor-1alpha on transcription of survivin in non-small cell lung cancer

Abstract

Background: 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.

Methods: 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.

Results: 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.

Conclusion: 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.

Figure 1

Expression of survivin and HIF-1α in NSCLC and benign lung disease tissues. Survivin and HIF-lα protein were detected and localised within paraffin-embedded human lung tissue using immunohistochemistry. A and B represent the negative expression of survivin protein and HIF-1α protein, respectively, in benign lung disease tissues. C and D represent the positive expression (arrow) of survivin protein and HIF-1α protein, respectively, in NSCLC,. E: The graph shows the statistical results. 81.60% (98/120) of lung cancer tissue samples were positive for survivin staining, and 58.33% (70/120_) of lung cancer tissue samples were positive for HIF-1α staining. ** p < 0.01.

Figure 2

Hypoxia induces expression of HIF-1α and survivin. A549 cells were cultured in 10% FBS medium under hypoxic or normoxic conditions for 24h. The relative levels of survivin (A) and HIF-1α (B) to GAPDH mRNA were determined by quantitative real time, reverse transcription-PCR. C: The expression of survivin and HIF-1α protein in A549 cells following HIF-1α-siRNA treatment as detected by Western blot analysis. D: The graph shows the statistical results of relative expression level of survivin and HIF-1α to β-actin protein. Data are given as means ± SD, n = 3, ** p < 0.01.

Figure 3

Site directed mutagenesis of the HIF-1α binding site on the survivin promoter decreases transcription activity of the survivin promoter. A: Nucleotide sequence of the survivin promoter. The putative binding sites for transcription factor are boxed. The GTGC sequence in -19 ~ -16 bp of survivin promoter was changed to AGC by mutation. B: A549 cells were transfected with pGL3-Basic without promoter (negative control), pGL3-SVP-229-luc (mutant plasmid), and pGL3-SVP-230-luc (normal plasmid). The relative activity of survivin promoter was analyzed by luciferase assay. The graph shows the statistical results. Data are given as means ± SD, n = 3, ** p < 0.01.

Figure 4

Decreased HIF-1α expression leads to decreased survivin expression in A549 cells. Cells were cultured in 10% FBS medium overnight, followed by treatment with HIF-1α-siRNA for 48 h. Total RNAs were isolated and analyzed by quantitative, real time, reverse transcription-PCR to determine the changes of survivin (A) and HIF-1α (B) mRNA. The relative levels of survivin and HIF-1α mRNA are expressed as a ratio of the amount of survivin (A) or HIF-1α (B) PCR products to the amount of GAPDH PCR product. C: The expression of survivin and HIF-1α protein in A549 cells following HIF-1α-siRNA treatment as detected by Western blot analysis. The relative expression levels of HIF-1α (D) and survivin (E) protein is expressed as a ratio of the amount of survivin or HIF-1α protein to the amount of β-actin protein. Data are given as means ± SD, n = 3, ** p < 0.01. Data are given as means ± SD, n = 3, ** p < 0.01.