01). Another HIF-1α binding site, located at -166 bp~-163 bp of the survivin

core promoter, was also mutated, but there was no BIBW2992 purchase relative difference in transcriptional activity between the normal and mutated binding site promoter constructs. 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 BMS202 in vitro 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. Decreased HIF-1α expression leads to decreased survivin expression in A549 cells A549 cells were treated with dsRNA (siRNA) targeted to HIF-1α mRNA and the expression levels of HIF-1α and survivin mRNA, and protein in were detected. As shown in Fig. 4, the mRNA and protein expression levels of HIF-1α and survivin in A549 cells significantly decreased after the treatment with HIF-1α siRNA as compared with negative control siRNA ASP2215 purchase and untreated controls (p < 0.05). Figure 4 Decreased HIF-1α expression leads to decreased survivin expression

in A549 cells. Cells were cultured in 10% FBS medium overnight, Lck 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. Discussion Apoptosis has negatively regulates the occurrence and development of tumors and prevents the rapid growth of tumor cells. Apoptosis is co-regulated by apoptosis-promoting factor and apoptosis-inhibiting factors (such as members of the IAP family of proteins) [22, 23]. Survivin, the smallest protein of IAP family, is rarely expressed in differentiated tissues and highly express in 75 ~ 96% of tumor tissues [4]. In this study, we found that survivin was expressed in 81.6% of NSCLC tissues, and not expressed in tissues from patients with benign lung diseases.