Figure 3: Drosha is downregulated by the ETS1/ELK1 complex under hypoxic conditions. | Nature Communications

Figure 3: Drosha is downregulated by the ETS1/ELK1 complex under hypoxic conditions.

From: Hypoxia-mediated downregulation of miRNA biogenesis promotes tumour progression

Figure 3

(a) Relative luciferase reporter activity for the Drosha promoter region under hypoxic conditions. β-Actin was used as a control. (b) Protein expression of ETS1 and ELK1 under hypoxic conditions in various cell lines. (c) Luciferase reporter activity for the wild-type (WT) Drosha promoter region and the ETS1 or ELK1 binding site–mutant Drosha promoter region under hypoxic conditions. (d) Drosha mRNA expression levels after ETS1, ElK1 and ETS/ELK1 siRNA gene knockdown under hypoxic conditions. (e) Anti-ETS1, anti-ELK1 and anti-POL II chromatin immunoprecipitation assay results showing fold enrichment of ETS1, ELK1 and POL II binding to the Drosha promoter region in A2780 cells. Rabbit IgG was used as a control and real-time PCR was used to quantitate the fold enrichment. (f) Effect of hypoxia on Drosha promoter methylation assessed by bisulfite conversion and methylation-specific PCR. The threshold cycle numbers obtained from samples with specific primers for unmethylated (UM) and methylated (M) sequences of the same promoter region of Drosha are shown. (g) mRNA and protein expression of Drosha in mouse tumour samples treated with B-20 (anti-VEGF antibody) and azacitidine. Scale bar, 200 μm. (h) Immunoprecipitation of hypoxia samples against ETS1 and ELK1 antibodies, probed for corresponding binding proteins ARID4B (ETS1) and HDAC1 (ELK1). (i) Anti-ARID4B and anti-HDAC1 chromatin immunoprecipitation assay results showing fold enrichment of ARID4B and HDAC1 binding to the Drosha promoter region in A2780 cells.(j) Aggregate tumour mass from tumours in the mouse orthotopic ovarian cancer model treated with control, ETS1, ELK1 and combination siRNAs. (k) Average mRNA expression of Drosha, ETS1 and ELK1 in the same tumour samples. All images shown are representative and data are presented as mean±s.e.m. of n≥3 experimental groups. **P<0.01, ***P<0.001 (Student t-test).

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