Effect Of HIF-1a MiRNA Interference On Survivin Expression In A549Cells

Object: To investigate the effect of hypoxia inducible factor-1α(HIF-1α) silence on theproliferation, apoptosis and immigration of A549cells as well as survivin expression,discovering the transcriptional regulation of survivin by HIF-1α in Non-Small CellLung Cancer (NSCLC). Methods: The pre-miRNA interference plasmid of HIF-1α for4sites (1170~1191,1207~1228,647~668,210~231) were successfully constructed.Following stable transfection of pcDNATM6.2-GW/EmGFP HIF-1α into lungadenocarcinoma cell line A549with LipofectamineTM2000, positive clone was selectedusing blasticidin. Then, RT-PCR and Western blot assay was performed to examine theexpression of HIF-1α and survivin selecting the cells with most interferencing effectunder normoxia or hypoxia respectively. Finally, cell proliferation, apotosis andmigration were measured by CCK8, flow cytometry (FCM) and transwell chambersunder normoxia and hypoxia, Results: The interference plasmid was verified bysequencing. Transfection of A549cells with HIF-1α miRNA resulted in decrease in thelevel of HIF-1α and Survivin protein as well as mRNA, which was further strengthenedunder hypoxia relative to normoxia. The RNAi plasmids capable of mostly effectivelyinhibiting HIF-1α expression (oligonucleotide targeted against the647-to668-bpregion) were selected for later use. CCK8assay showed transfection of cell with HIF-1αmiRNA significantly inhibited cell proliferation under hypoxia. The apoptotic ratio oftransfected cells under hypoxia was also higher than that of control. Interestingly, thecells migration was substantially suppressed by HIF-1α silence under both normoxiaand hypoxia. Conclusion: The recombinant plasmid HIF-1α miRNA effectively inhibitsthe expression of HIF-1α as well as survivin in A549cells. HIF-1α is able to activatesurvivin transcription, thereby inhibiting cell proliferation, inducing apoptosis underhypoxia in lung cancer.