P53-induced microRNA-107 inhibits HIF-1 and tumor angiogenesis

Abstract

The pathway involving the tumor suppressor gene TP53 can regulate tumor angiogenesis by unclear mechanisms. Here we show that p53 regulates hypoxic signaling through the transcriptional regulation of microRNA-107 (miR-107). We found that miR-107 is a microRNA expressed by human colon cancer specimens and regulated by p53. miR-107 decreases hypoxia signaling by suppressing expression of hypoxia inducible factor-1beta (HIF-1beta). Knockdown of endogenous miR-107 enhances HIF-1beta expression and hypoxic signaling in human colon cancer cells. Conversely, overexpression of miR-107 inhibits HIF-1beta expression and hypoxic signaling. Furthermore, overexpression of miR-107 in tumor cells suppresses tumor angiogenesis, tumor growth, and tumor VEGF expression in mice. Finally, in human colon cancer specimens, expression of miR-107 is inversely associated with expression of HIF-1beta. Taken together these data suggest that miR-107 can mediate p53 regulation of hypoxic signaling and tumor angiogenesis.

Fig. 1.

p53 increases miR-107 expression. (A) Human PANK1 gene structure. miR-107 is encoded within an intron in the PANK1 gene. The gene encoding miR-107 is shown in black; the PANK1 gene also includes untranslated exons (white) and translated exons (gray). (B) Exposure to etoposide for 24 h increases miR-107 expression in HeLa cells by Northern blotting. (C) Exposure to etoposide for 24 h increases miR-107 only in HCT116 cells that express p53 by quantitative real-time PCR. (D) PANK1/pri-miR-107 but not PANK2/pri-miR-103–2 or PANK3/pri-miR-103–1 behaves like a p53 target gene. HCT116(WT) and HCT116(p53 KO) cells were exposed to Adriamycin for 24 h before transcript abundance was measured by quantitative PCR and normalized to U6. (E) Schematic diagram of the various PANK1 promoter reporter constructs containing a putative p53 binding element cloned into the pGL3 basic luciferase reporter plasmid. (F) Exposure to DNA-damaging agents for 24 h increases miR-107 promoter activity in HCT116 cells transfected with miR-107 reporter vector (n = 3 ± SD; *, P < 0.05). (G) Etoposide transactivates a miR-107 reporter vector containing a p53 binding site but fails to transactivate a shorter vector lacking the p53 binding site (n = 3 ± SD; *, P < 0.05 vs. control). (H) Etoposide fails to transactivate a miR-107 reporter vector with a specific mutation in the p53 binding site at −1,811 bp (n = 3 ± SD; *, P < 0.05 vs. control). (I) p53 interacts with miR-107 promoter in the region of the p53 binding site as demonstrated by ChIP. (J) p53 interacts with miR-107 promoter in the region of the p53 binding site as shown by quantitative PCR analysis of ChIP (n = 3 ± SD; *, P < 0.05)

Fig. 2.

HIF-1β is a target of miR-107. (A) Manipulation of miRNA-107 in HCT116. Transfection of HCT116 cells with anti-sense miR-107 decreases endogenous miR-107 levels (± SD, *P < 0.05) (Left). Transfection of HCT116 cells with pre-miR-107 increases miR-107 levels (Right). (B) Transfection of HCT116 cells with pre-miR-107 suppresses HIF-1β protein levels as shown by immunoblotting. (C) Quantification of dose-dependent suppression of HIF-1β protein levels by miR-107 in HCT116 cells by densitometry (n = 3 ± SD; *, P < 0.05). (D) HCT116 cells were transfected with pre-miR-control or pre-miR-107 and exposed to normoxia or hypoxia, and the levels of HIF-1β and HIF-1alpha were probed by immunoblotting. miR-107 affects only HIF-1β expression, not HIF-1α. (E) Transfection of human colon cancer cell lines HCT116 and SW480 or the human epithelial cancer HeLa cell line with antisense (AS) to miR-107 slightly decreases HIF-1β protein levels. (F) Pre-miR-107 decreases HIF-1β expression in HCT116 cells, but other miRNA do not. (G) The HIF-1β 3′ UTR contains a binding site for miR-107. (Top) The HIF-1β 3′ UTR contains a potential miR-107 binding site with an exact 8-nt match. (Middle) Mutation of the miR-107 binding site in the HIF-1β 3′ UTR. (Bottom) Schematic of reporter vector containing luciferase followed by the HIF-1β 3′UTR. (H) miR-107 decreases transactivation of HIF-1β 3′ UTR. HCT cells were transfected with a reporter vector containing luciferase followed by 1,000 bp of the 3′ UTR of HIF-1β, containing either a WT UTR or a mutant UTR lacking the miR-107 binding site. Cells were transfected with control siRNA or pre-miR-107, and luciferase activity was measured (n = 3 ± SD; *, P < 0.05 vs. mutant binding site).

Fig. 3.

miR-107 regulates hypoxia signaling. (A) Hypoxia (1% O₂) for 24 h or the hypoxia mimetic DFX for 24 h increases secretion of VEGF protein from HCT116 cells as measured by ELISA (n = 3 ± SD; *, P < 0.05 vs. control). (B) Exogenous miR-107 decreases secretion of VEGF from HCT116 cells transfected with siRNA control oligo or pre-miR-107 and then treated with DFX for 24 h (n = 3 ± SD; *, P < 0.05). (C) Endogenous miR-107 decreases secretion of VEGF from HCT116 cells transfected with siRNA control oligo or anti-sense (AS)-miR-107 and then treated with DFX for 24 h (n = 3 ± SD; *, P < 0.05). (D) miR-107 affects cellular stimulation of endothelial proliferation. HCT116 cells were transfected with control or pre-miR-107 and exposed to hypoxia for 16 h. Then the conditioned media were transferred to endothelial cells. Human umbilical vein endothelial cell proliferation was measured after 48 h by the MTT assay (n = 3 ± SEM; *, P < 0.05). (E) Manipulation of HIF-1β in HCT116 cells. HIF-1β siRNA transfection decreases HIF-1β protein expression in HCT116 cells (Upper rows). HIF-1β expression vector (pCMV-HIF-1β) transfection increased HIF-1β protein expression in HCT116 cells (Lower rows). (F) HIF-1β rescues cells from miR-107 inhibition of hypoxia signaling. Overexpression of HIF-1β in HCT116 cells restores DFX stimulation of VEGF protein secretion measured by ELISA (n = 3 ± SD; *, P < 0.05 for HIF-1β vs. control vector). (There is no significant difference between pre-miR-scr + DFX and pre-miR-107 + DFX in cells transfected with HIF-1β vector. The cDNA encoding HIF-1β lacks an miR-107 response element, so pre-miR-107 does not affect plasmid expression of HIF-1β.)

Fig. 4.

miR-107 decreases tumor volume and vessels. (A) Schematic of LV-GFP-miR-107. (B) GFP fluorescence of HCT116 cells transduced with control media (Left) or with lentivirus expressing GFP (Right) analyzed by FACS. (C) LV-GFP-miR-107-transduced cells (solid bars) contain 10-fold more miR-107 than LV-GFP–infected cells (white bars) by quantitative real-time PCR (n = 3–10 ± SD; *, P < 0.05). (D) miR-107 decreases tumor volume. Nude mice were injected with 2 million HCT116 cells after transduction with lentiviral vectors. Four different HCT116 cell types were injected: HCT116(WT) and LV-GFP; HCT116(WT) and LV-miR-107; HCT116(p53 KO) and LV-GFP; and HCT116(p53 KO) and LV-miR-107. Tumor dimensions were measured for 31 d. Expression of miR-107 decreases the size of HCT116(WT) and HCT116(P53 KO) tumors (n = 10; *, P < 0.05 vs. LV-miR-107). (E) miR-107 decreases the density of blood vessels in tumors, as assessed by immunohistochemistry for VWF. (F) miR-107 decreases the number of tumor blood vessels, as measured by counting the number of vessels per high-power field for 12–15 fields for three tumors in each of the four groups (n = 3 ± SD; *, P < 0.001). (G) miR-107 decreases expression of the hypoxia-regulated gene VEGF. Quantitative real-time PCR was used to analyze expression of VEGF in tumors from nude mice implanted with HCT116 cells transduced with lentiviral vectors (n = 3–10 ± SD; *, P < 0.05). (H) miR-107 is inversely related to VEGF expression in human colon cancer. We measured miR-107 expression and VEGF expression by quantitative real-time PCR in 32 specimens of colon cancer harvested from patients at the Johns Hopkins University School of Medicine. We grouped the 32 human colon cancer specimens into the 16 specimens with the lowest expression of miR-107 and the 16 specimens with the highest expression of miR-107 (n = 16 ± SD; *, P < 0.0001). VEGF expression is higher in the low miR-107 group than in the high miR-107 group (n = 16 ± SD; *, P < 0.03). (I) Schematic for miR-107 regulation of hypoxia signaling.