HIF-1α knockdown by miRNA decreases survivin expression and inhibits A549 cell growth in vitro and in vivo

Abstract

The present study examined the downregulation of survivin expression by hypoxia-inducible factor-1α (HIF-1α) miRNA and its effect in the inhibition of A549 cell growth in vitro and in vivo. Survivin expression, apoptosis, proliferation and migration under normoxic and hypoxic conditions were assessed by standard methods. Cotransfection and chromatin immunoprecipitation were used to observe the effects of HIF-1α on survivin transcription. HIF-1α knockdown in A549 cells were injected into nude mice to examine survivin expression and suppression of tumorigenicity. Transfection of A549 cells with HIF-1α miRNA led to decreased expression of HIF-1α and survivin mRNA and protein. Survivin overexpression is mediated by HIF-1α by direct binding to a putative binding site in the survivin core promoter. HIF-1α-miRNA induced apoptosis and inhibited proliferation of A549 cells under hypoxic, but not normoxic, conditions, whereas transfection by survivin expression vectors partly rescued the apoptotic phenotype and revived cell proliferation under hypoxic conditions. However, cell migration was substantially suppressed by HIF-1α silencing under normoxic and hypoxic conditions. After A549 cells were xenografted in nude mice, survivin expression in mice treated with HIF-1α miRNA was downregulated, and tumor growth was significantly inhibited. Silenced HIF-1α gene expression induced apoptosis and suppressed growth of A549 cells by downregulating survivin expression in vitro and in vivo. Our results also provide a basis to target the HIF-1α pathway in lung cancer therapy.

Figure 1

HIF-1α expression knocked down by miRNA plasmids in A549 cells under hypoxia. (A) Western blot analysis of HIF-1α and survivin protein expression using total protein extracted from cells. (B) HIF-1α and survivin gene expression were measured by RT-PCR using total RNA isolated from cells. Data are shown as means ± SD, n=3. ^*P<0.05.

Figure 2

Mechanism of HIF-1α-activated survivin gene expression. Cultures were cotransfected with pGL3-SVP-230 and either HIF-1α or control vector in A549 cells under normoxia (A) and hypoxia (B). Relative survivin promoter activity was assayed by luciferase. A549 cells were transfected with HIF-1α or empty vector; ChIP assay then confirmed HIF-1α directly binding to survivin promoter under hypoxia (C) and normoxia (D). Prior to immunoprecipitation, input was used as an internal control; mouse IgG was used as negative control. Data represent means ± SD (n=3). ^*P<0.05, ^**P<0.01, vs. control.

Figure 3

CCK-8 assay. Longitudinal axis shows 450-nm OD microplate value for each well to determine A549 cell viability, for untreated, Scrambled, miRNA plasmid-transfected and survivin plasmid + miRNA groups. Latitudinal axis shows days after cells were treated with or without CoCl₂ in a 96-well plate under normoxia (A) and hypoxia (B). Data are shown as means ± SD; n=3. ^*P<0.05.

Figure 4

Apoptosis detected by flow cytometry. Apoptosis rate in the HIF-1α-miRNA group was significantly higher than in groups treated with Scrambled, untreated or survivin plasmid + miRNA (P<0.05) under hypoxia; however, the effect of HIF-1α-miRNA on apoptosis under normoxia did not differ significantly between the Scrambled and untreated groups (data not shown).

Figure 5

Invasiveness of A549 cells shown by Transwell assay under normoxia and hypoxia. Invasiveness was less in HIF-1α miRNA-transfected cells, as shown by fewer cells invading the lower poly-carbonic membrane surface under normoxia compared with controls (A); invasiveness of treated cells was even less under hypoxia (B). Data are given as means ± SD; n=3. ^*P<0.05; ^**P<0.01.

Figure 6

HIF-1α-miRNA resulted in significantly inhibited tumor growth. Mice were inoculated subcutaneously with A549 cells and treated with HIF-1α-miRNA or Scrambled RNA. (A) Growth curves show that tumors of the HIF-1α-miRNA group grew slower than in the untreated and Scrambled groups. (B) Representative images show tumor volume of the HIF-1α-miRNA group was less than in the untreated and Scrambled groups.

Figure 7

Knocked down HIF-1α expression by miRNA plasmids in transplanted tumor tissue. (A) HIF-1α and survivin gene expression were measured by RT-PCR using total RNA isolated from transplanted tumor tissue. (B) Western blot analysis of HIF-1α and survivin protein expression using total protein extracted from transplanted tumor tissue. Data are given as means ± SD; n=3. ^*P<0.05.

Figure 8

Apoptosis in 3 transplanted tumor tissues, examined by TUNEL assay. (A) Representative images show apoptosis of tumors in HIF-1α-miRNA, Scrambled and untreated groups. (B) Percentage of TUNEL⁺ cell nuclei calculated relative to total number of cell nuclei. Data are given as means ± SD; n=3. ^*P<0.05.