A microRNA signature of hypoxia

Abstract

Recent research has identified critical roles for microRNAs in a large number of cellular processes, including tumorigenic transformation. While significant progress has been made towards understanding the mechanisms of gene regulation by microRNAs, much less is known about factors affecting the expression of these noncoding transcripts. Here, we demonstrate for the first time a functional link between hypoxia, a well-documented tumor microenvironment factor, and microRNA expression. Microarray-based expression profiles revealed that a specific spectrum of microRNAs (including miR-23, -24, -26, -27, -103, -107, -181, -210, and -213) is induced in response to low oxygen, at least some via a hypoxia-inducible-factor-dependent mechanism. Select members of this group (miR-26, -107, and -210) decrease proapoptotic signaling in a hypoxic environment, suggesting an impact of these transcripts on tumor formation. Interestingly, the vast majority of hypoxia-induced microRNAs are also overexpressed in a variety of human tumors.

FIG. 1.

Coordinated hypoxic changes of microRNA expression in colon and breast cancer cell lines. Cluster analysis of four cell lines according to the expression of microRNAs upregulated by hypoxia in at least two cell lines. Expression data were normalized to expression at time zero.

FIG. 2.

Confirmation of HRM induction by hypoxia by Northern analysis or quantitative RT-PCR. (a) Northern blotting confirmation of miR-210 induction under hypoxia (the mature form is indicated). Lanes NOR, 6HY, and 30HY show results under normoxia and at 6 h and 30 h under hypoxia, respectively. An ethidium bromide-stained gel picture is shown as a loading control. (b) Quantitative RT-PCR confirmation of HRM induction by 24-hour hypoxia (H) compared with HRM induction for normoxic controls (N). Bars indicate means from two independent experiments. I bars indicate standard deviations.

FIG. 3.

Distribution of random 23-microRNA groups (samples) based on HIF1_Q3 (a) or HIF1_Q5 (b) binding sites. The arrow indicates where the experimental data (HRMs) fall within the random sample population, with the corresponding P value.

FIG. 4.

Effect of HIF on specific microRNA expression. The impact of exogenous constitutively active HIF-1α (HIF1P/A) and HIF-2α (HIF2P/A) subunits on miR-103, -210, and -213 expression was determined by quantitative RT-PCR in HT29 (a) and MCF7 (b) cells. The control was the pcDNA3.1 empty vector (PC). Bars indicate means from two independent experiments. I bars indicate standard deviations.

FIG. 5.

Direct effect of HIF in the up-regulation of select HRMs. Relative luciferase activities of HRM promoter reporter constructs in HT29 cells. (a) miR-24-1 and -181c promoters in a pGL3 context; (b) miR-26b fragment in a pGL3-tk context. The constructs were cotransfected with constitutively active HIF-1α (HIF1P/A), HIF-2α (HIF2P/A), or the empty vector pcDNA3.1 (PC) and incubated under normoxia (NOR). The effect of hypoxia (HYP) on the reporter is also shown. Bars indicate means from three independent experiments. I bars indicate standard deviations.

FIG. 6.

Direct recruitment of HIF on the miR-210 and miR-26 promoters under hypoxia. Chromatin was immunoprecipitated from HT29 cells using a HIF-1α antibody or an IgG control, and the enriched genomic fragment was amplified using primers spanning the candidate HREs located at positions −1720 and −1822 (210-A); −1166 (210-B) of the miR-210 promoter; or −2860 for the miR-26a-2 promoter (26-A). A fragment of the miR-130b promoter was used as a negative control.

FIG. 7.

Antiapoptotic effect of select microRNAs under hypoxia. Blockade of miR-26, -107, and -210 with antisense inhibitors leads to an increased apoptotic response in three independent experiments (each performed in triplicate). In contrast, an excess of sense microRNAs decreases the apoptotic response. The dotted line represents the apoptotic caspase-3/7 baseline activity in response to negative-control microRNA under hypoxia (P, precursor [sense]; A, antisense). (b) Northern blot confirmation of efficient transduction or blockade of miR-210 in MCF7 cells. MCF7 cells were transfected with the precursor or antisense miR-210 or the scramble control (SCR). U6 snRNA is shown as the loading control.