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. 2014 Jun 26;5(6):e1301.
doi: 10.1038/cddis.2014.240.

Hypoxia-induced miR-424 decreases tumor sensitivity to chemotherapy by inhibiting apoptosis

D Zhang  1 , Z Shi  2 , M Li  3 , J Mi  2
Affiliations

Hypoxia-induced miR-424 decreases tumor sensitivity to chemotherapy by inhibiting apoptosis (VSports)

D Zhang et al. Cell Death Dis. .

Abstract

Chemotherapy resistance of tumor cells is a big challenge. Adaption to hypoxia is an essential cellular response that is controlled by the master oxygen-sensitive transcription factor HIF1 (hypoxia-inducible factor 1). The mechanism by which tumor cells acquire resistance to chemotherapy under hypoxic conditions is not fully understood. In this study, we found that hypoxia induces miR-424 expression and that miR-424 in turn suppresses the level of PDCD4 protein, a tumor suppressor that is involved in apoptosis, by targeting its 3' untranslated region. Functionally, miR-424 overexpression decreases the sensitivity of cancer cells (HCT116 and A375) to doxorubicin (Dox) and etoposide. In contrast, the inhibition of miR-424 enhanced apoptosis and increased the sensitivity of cancer cells to Dox. In a xenograft tumor model, miR-424 overexpression promoted tumor growth following Dox treatment, suggesting that miR-424 promotes tumor cell resistance to Dox. Furthermore, miR-424 levels are inversely correlated with PDCD4 expression in clinical breast cancer samples. These results suggest that miR-424 is a potential molecular target for tumor therapy. VSports手机版.

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Figures

Figure 1
Figure 1
Hypoxia increases miR-424 expression in cancer cells. (a) Hypoxia increases miR-424 expression in melanoma A375 cells. A selected heatmap of microRNA profiling, which was analyzed by deep sequencing in melanoma A375 cells, is shown for different time points under hypoxic conditions. The color from green to dark red indicated the expression level of miRNAs, from low to high. (b). Hypoxia increases the level of mature miR-424 in different cancer cell lines. The level of miR-424 was individually analyzed using quantitative PCR analysis in A375, U251, and HCT116 cells, which were treated with 1% O2, 2 mM DMOG, or 40 μM H2O2. (c) Hypoxia increases miR-424 expression in different cancer cell lines. The level of pri-miR-424 was analyzed using quantitative PCR in the A375, U251, and HCT116 cancer cell lines treated as above. (d) The protein level of HIF1α increased under hypoxia. The A375, HCT116, and U251 cells were treated with 1% O2, 2 mM DMOG, or 40 μM H2O2; the protein level of HIF1α was evaluated by western blot analysis. (e) HIF1α spefically binds the putative HRE site in the promoter of miR-424. Chromatin IP was performed in hypoxia-induced A375 cells (12 h) using antibody against HIF1α, or IgG, and analyzed by qPCR. MiR-424 occupancy by HIF1α was significantly enriched compared to IgG. (f) 293 T cells were cotransfected with pGL3p control, pGL3p-wt miR-424 HRE (HRE wt), or pGL3p-mutant miR-424 HRE (HRE mut) and pSV-Renilla and were exposed to 20% or 1% O2 for 12 h. The ratio of Fluc:Rluc activity was normalized to control at 20% O2. The miR-424 HRE significantly increased Fluc activity in hypoxic condition
Figure 2
Figure 2
The overexpression of miR-424 suppresses the apoptosis of tumor cells induced by anti-cancer chemicals. (a and b) Cleaved PARP and cleaved caspase-3 were detected using western blot analysis in HCT116 and A375 cells, which were transfected with a scrambled oligo or a miRNA-424 mimic. The final concentration of doxorubicin was 1 nM. (c and d) The annexin V-positive population of HCT116 or A375 cells was analyzed using flow cytometry. The final concentration of etoposide was 100 μM or 5 μM. (e) Cleaved PARP and cleaved caspase-3 levels were detected using western blot analysis in A375 cells that were transfected with a scrambled oligo or the miRNA-424 inhibitor. (f and g) The annexin V-positive populations of HCT116 or A375 cells were analyzed using flow cytometry. The final concentration of etoposide was 100 μM and 5 μM, respectively. (h) The survival ratio was analyzed using the CCK8 assay in HCT116 cells treated with doxorubicin (10 nM). HCT116 cells were transfected with the miRNA-424 mimic or inhibitor
Figure 3
Figure 3
miR-424 regulates PDCD4 expression. (a) Proteins regulating apoptosis were predicted to be targets of miR-424. (b) This sequence in the 3′UTR of PDCD4 was predicted to bind to miR-424. The nucleotides in red were mutated to their complementary nucleotides. (c) 3′UTR luciferase assay showing the repression of wild-type UTR or mutant UTR following transfection of the miR-424 mimic or scrambled miRNA (*P<0.05; #P>0.05). (d) miR-424 overexpression inhibits the protein level of PDCD4 in A375 and fibroblast cells, which were evaluated using western blot analysis. (e) miR-424 knockdown increased the protein level of PDCD4 in A375, U251, and HCT116 cells. (f) The miR-424 mimic inhibited the mRNA level of PDCD4 in A375 and HCT116, as analyzed by quantitative PCR. (g) The miR-424 inhibitor increased the mRNA level of PDCD4 in A375 and HCT116 cells
Figure 4
Figure 4
miR-424 decreases the sensitivity of tumor cells to doxorubicin in vivo. (a) Dox (2 mg/ml) was administrated to xenograft mice in the drinking water. The photo is a representative image of the xenograft tumors. (b) The tumor growth rate were analyzed between the four groups. (c) The TUNEL-positive cells were analyzed in the tumor sections (original magnification, 20 × ); the percentage of TUNEL-positive cells in this four groups was quantified. (d) Dox treatment did not change PDCD4 expression. The expression of PDCD4 in xenograft tumors was analyzed by western blot. (e) Correlation analysis of miR-424 and PDCD4 expression in clinical breast cancer samples using the Pearson correlation (R=−0.48035, P<0.01, n=257)
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References

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