MicroRNA-17, 20a Regulates the Proangiogenic Function of Tumor-Associated Macrophages via Targeting Hypoxia-Inducible Factor 2α

<div><p>Tumor-associated macrophages (TAMs) constitute a major component of the leukocyte infiltrate of most solid tumors, and they usually exhibit a proangiogenic phenotype which facilitates tumor growth in most circumstances. However, the precise mechanisms regulating the proangiogenic properties of TAMs remain largely unclear. In the present study, we found that the expression of hypoxia-inducible factor 2α (HIF-2α) was significantly up-regulated in macrophages from tumor tissues of several solid tumors. Macrophages exposed to tumor cell line derived-culture supernatants (TSN) also expressed high levels of HIF-2α in vitro, without a requirement for hypoxia. We identified miR-17 and miR-20a as the key regulators of HIF-2α expression in TAMs, and autocrine IL-6 played an important role in mediating the expression of miR-17, miR-20a, and thereafter HIF-2α in TAMs. Furthermore, the elevated HIF-2α in TAMs stimulated transcription of a set of proangiogenic genes such as VEGFA and PDGFB, which might in turn contribute to the angiogenic process within tumors. Our data provide evidence in support of the critical role of HIF-2α in the proangiogenic activity of TAMs and also reveal a novel mechanism by which miRNAs regulate TAM functions through modulation of HIF-2α expression under non-hypoxic conditions.</p></div

HIF-2α modulates proangiogenic gene expression in TAMs.

<p>(A) CD14⁺ cells were isolated from non-tumoral (Non-) or intratumoral (Intra-) tissue of seven HCC patients. (B) Healthy PBMC-derived CD14⁺ cells were treated with medium alone or HepG2 TSN for 7 days. (C, D) Healthy PBMC-derived CD14⁺ cells were treated with HepG2 TSN and transfected with NC, si-HIF-2α (C), or miR-17 and miR-20a mixture (D). Levels of VEGFA and PDGFB mRNA expression in these cells were analyzed by qPCR. Upper panel of C shows the knockdown efficiency of si-HIF-2α. All results are normalized to ACTB and presented as fold-changes. All data shown are representative of at least four separate experiments. *<i>P</i><0.05, **<i>P</i><0.01 compared with the indicated groups.</p

Role of IL-6 in downregulation of miR-17 and miR-20a and upregulation of HIF-2α expression in TAMs.

<p>(A) Level of IL-6 mRNA in CD14⁺ cells isolated from non-tumoral (Non-) or intratumoral (Intra-) tissue of seven HCC patients was analyzed by qPCR. Results are presented as fold-changes. (B) Healthy PBMC-derived CD14⁺ cells were treated with medium alone or HepG2 TSN for 24h. IL-6 concentrations in the culture supernatants were determined by ELISA. (C, D, E) Healthy PBMC-derived CD14⁺ cells were treated HepG2 TSN and transfected with NC or si-IL-6R (C). Healthy PBMC-derived CD14⁺ cells were treated with medium, TCM, TCM with anti-IL-6, or TCM with IgG control for 7 days (D). Healthy PBMC-derived CD14⁺ cells were treated with medium alone or recombinant IL-6 for 7 days (E). MiR-17 and miR-20a in these cells were analyzed by qPCR. Expression of HIF-2α was determined by Western blotting. qPCR data were presented as fold-changes and normalized to ACTB for mRNA expression or RNU6B for miRNAs expression. Values represent the mean ± SEM for A and mean ± SD for the others. All data shown are representative of at least four separate experiments. *<i>P</i><0.05, **<i>P</i><0.01 compared with the indicated groups.</p

Upregulation of HIF-2α expression in TAMs.

<p>(A) Adjacent sections of paraffin-embedded HCC tissue (n = 9) were stained with an anti-HIF-2α or anti-CD68 antibody. (B) HIF-2α expression in macrophages from frozen sections of HCC tissue (n = 6) was analyzed by confocal microscopy. HIF-2α, red; CD68, green; DAPI, blue. (C) Level of HIF-2α protein in CD14⁺ cells isolated from non-tumoral (Non-) or intratumoral (Intra-) tissue of 4 HCC patients was analyzed by immunoblotting. (D) Healthy PBMC-derived monocytes were treated with medium alone (Med) for 7 days, or with HepG2 TSN under normoxic condition (HepG2 TSN) for 7 days, or with medium alone for 6 days and then exposed to 1% O₂ for another 24 h (hypoxia). Expression of HIF-2α and HIF-1α was determined by Western blotting. This data shown are representative of four separate experiments.</p

MiR-17 and miR-20a regulate HIF-2α expression at the post-transcriptional level in TAMs.

<p>(A, B) CD14⁺ cells were isolated from non-tumoral (Non-) or intratumoral (Intra-) tissues of seven HCC patients (A). Healthy PBMC-derived CD14⁺ cells were treated with medium alone or HepG2 TSN for 7 days (B). Levels of HIF-2α mRNA, miR-17 and miR-20a in these cells were analyzed by qPCR. Data were presented as relative level and normalized to ACTB for mRNA expression or RNU6B for miRNAs expression. (C) Pairing of miR-17 and miR-20a with three HIF-2α 3′UTR regions (nucleotides 170–176, 816–822 and 1500–1506 of human HIF-2α 3′UTR), and mutations introduced into the luciferase reporter construct are shown. (D, E) Luciferase activity of HepG2 cells transfected with the indicated reporters was analyzed. pRL-CMV expressing <i>Renilla</i> luciferase was cotransfected as an internal control and the luciferase activity of each sample was normalized to this. The luciferase activity of the NC or anti-miR-C transfectant was assigned an arbitrary value of 1. (F) Healthy PBMC-derived CD14⁺ cells were treated with medium alone and then transfected with NC or anti-miR-17 and anti-miR-20a mixture (anti-miRNAs), or with HepG2 TSN and then transfected with NC or miR-17 and miR-20a mixture (miRNAs). HIF-2α expression was analyzed by Western blotting. Values represent the mean ± SEM for A and mean ± SD for the others. All data shown are representative of at least four separate experiments. *<i>P</i><0.05, **<i>P</i><0.01 compared with the indicated groups.</p

Model of the novel mechanism regulating HIF-2α expression under non-hypoxic condition within tumor microenvironment.

<p>Soluble factor(s) derived from tumor cells induced the secretion of IL-6 by stimulating its transcription. Autocrine IL-6 triggered the down-regulation of miR-17 and miR-20a, which in turn released the suppression of HIF-2α and led to its accumulation under non-hypoxic condition. HIF-2α further promoted the transcription of proangiogenic genes such as VEGFA and PDGFB and contributed to tumor angiogenesis.</p