miR-210 is overexpressed in late stages of lung cancer and mediates mitochondrial alterations associated with modulation of HIF-1 activity

Abstract

Following the identification of a set of hypoxia-regulated microRNAs (miRNAs), recent studies have highlighted the importance of miR-210 and of its transcriptional regulation by the transcription factor hypoxia-inducible factor-1 (HIF-1). We report here that miR-210 is overexpressed at late stages of non-small cell lung cancer. Expression of miR-210 in lung adenocarcinoma A549 cells caused an alteration of cell viability associated with induction of caspase-3/7 activity. miR-210 induced a loss of mitochondrial membrane potential and the apparition of an aberrant mitochondrial phenotype. The expression profiling of cells overexpressing miR-210 revealed a specific signature characterized by enrichment for transcripts related to 'cell death' and 'mitochondrial dysfunction', including several subunits of the electron transport chain (ETC) complexes I and II. The transcript coding for one of these ETC components, SDHD, subunit D of succinate dehydrogenase complex (SDH), was validated as a bona fide miR-210 target. Moreover, SDHD knockdown mimicked miR-210-mediated mitochondrial alterations. Finally, miR-210-dependent targeting of SDHD was able to activate HIF-1, in line with previous studies linking loss-of-function SDH mutations to HIF-1 activation. miR-210 can thus regulate mitochondrial function by targeting key ETC component genes with important consequences on cell metabolism, survival and modulation of HIF-1 activity. These observations help explain contradictory data regarding miR-210 expression and its putative function in solid tumors.

Figure 1

Overexpression of miR-210 in NSCLC is linked to hypoxia induction in tumors. (a) Unsupervised hierarchical clustering analysis of 42 miRNAs in 20 NSCLC samples. Distance was measured using the Manhattan distance on the matrix of the log2 (ratio) and classification was performed using a complete agglomeration method. Histograms on the right of the panel represent miR-210, miR-31 and miR-451 modulation in three different stages of the disease. (b) Expression of miR-210 and the hypoxic index in 13 NSCLC samples. Histograms represent the log2 ratio of tumor versus normal adjacent tissues for miR-210 and for a hypoxic index corresponding to the average value of the 15 top-ranked genes (see Supplementary Table S3 for the list of the transcripts) of the common hypoxia metagene identified by Buffa et al. Similar results were obtained with the full list of this common hypoxia signature. (c) miR-210 expression was monitored by qPCR on RNA from A549 cells with or without exposure to 1% O₂ at indicated times. Mean±S.E.M. is representative of three independent experiments carried out in triplicate. ^***P<0.0005, ^**P<0.005, ^*P<0.05

Figure 2

miR-210 targets E2F3 and alters A549 cell viability. (a) A549 cells were transfected with 10 nM pre-miR-210, premiR-34a, pre-miR-Neg, si-Neg or si-E2F3 as a positive control. E2F3 protein levels were determined by western blotting on day 2 after transfection. HDAC1 expression was used as a loading control. (b) A549 cells were co-transfected with 10 nM pre-miR-Neg, pre-miR-210 or pre-miR-34a and different pSI-Check-2 constructs. Cells were harvested 2 days after transfection and the luciferase activities analyzed. All Renilla luciferase activities were normalized to the firefly luciferase activity. pSI-E2F3-1 and pSI-E2F3-2 correspond to distinct fragments of the E2F3 3′UTR containing putative miR-210 and miR-34a putative binding sites, respectively; pSI-check2 was used as an empty vector control. (c) The XTT cell viability assay was performed on days 1–5 after transfection of A549 cells with pre-miR-Neg, pre-miR-210, pre-miR-34a, si-Neg or si-E2F3. (d) The caspase-3/7 assay was carried out on A549 cells transfected under the same conditions as in (c). Results correspond to three independent experiments performed in duplicate. ^***P<0.0005, ^**P<0.005

Figure 3

Identification of miR-210 candidate targets using a transcriptomic approach. A549 cells were transfected with pre-miR-Neg, pre-miR-210, pre-miR-34a, as well as siE2F3 or a control siRNA (si-Neg) (n=2). RNA samples were harvested at 48 h after transfection and expression profiles were determined with pangenomic arrays. (a) Heatmap comparing the normalized log2 of the ratios between the signal in the different conditions and the pre-miR-Neg signal at 48 h after transfection. (b and c) Overrepresentation of miR-210 (b) and miR-34a (c) predicted targets according to TargetScan algorithm following transfection with pre-miR-210 and pre-miR-34a, respectively. Representation of miRNA predicted targets in the set of up- or downregulated genes was compared with the set of all expressed genes. For each miRNA, a fold enrichment value (horizontal axis) and an associated P-value (vertical axis) were calculated. (d) Histogram representing the enrichment for the presence of seed complementary hexamers in the 3′UTR of down- or upregulated transcripts following transfection with pre-miR-210, pre-miR-34a and si-E2F3. ^***P<0.0005

Figure 4

miR-210 targets SDHD and induces mitochondrial dysfunction. (a) Direct targeting of SDHD and NDUFA4 by miR-210 in vitro. A549 cells were co-transfected with 10 nM of pre-miR-Neg, pre-miR-210 or pre-miR-34a with pSI-Check-2 constructs containing the 3′UTR of SDHD (pSI-SDHD) or NDUFA4 (pSI-NDUFA4). Cells were harvested 2 days after transfection and luciferase activities were analyzed. (b) Effect of miR-210 expression on endogenous members of ETC complexes I and II. The A549 cell line was transfected with 10 nM pre-miR-Neg, pre-miR-210 or pre-miR-34a. SDHA, NDUFA9 and NDUFS7 protein levels were determined by western blotting on day 2 after transfection. Total ERK2 was used as a loading control. (c) Complex II activity was performed on A549 cells on day 3 after transfection with pre-miR-Neg or pre-miR-210. (d) Electron micrographs of the ultrastructure of mitochondria of A549 cells transfected with miR-Neg or miR-210. All data are representative of three independent experiments. ^**P<0.005, ^*P<0.05

Figure 5

SiRNAs directed against SDHD induce modifications in mitochondrial shape and apoptosis. (a) Mitochondrial ultrastructure in A549 cells transfected with pre-miR-Neg, pre-miR-210, si-Neg or two different si-SDHD alone or together and fixed in situ at 4 days after transfection. miR-210 and si-SDHD strongly modified mitochondrial structure (arrow). (b) XTT cell proliferation and (c) caspase-3/7 assays were performed on A549 cells transfected with pre-miR-Neg, pre-miR-210, si-Neg or si-SDHD. All data are representative of three independent experiments performed in triplicate. ^***P<0.0005, ^**P<0.005

Figure 6

miR-210 inhibition decreases cell survival and affects the phenotype of mitochondria in hypoxia. A549 cells were transfected with either pre-miR-Neg, pre-miR-210, a control anti-miR-159s LNA or anti-miR-210, before 72–96 h exposure to normoxia or 1% O₂. (a) miR-210 expression was monitored by qPCR on RNA from A549 cells with or without exposure to 1% O₂ (72 h). (b) Following exposure to normoxia or hypoxia (72 h), cells were re-plated (100 and 500 cells per well) and incubated during 11 days. Colonies were then fixed, stained, and the surviving fractions were calculated based upon the plating efficiency. (c) Quantification of the average number of cristae in cells fixed in situ 96 h following exposure to normoxia or hypoxia. Mean±S.E.M. is representative of two independent experiments carried out in triplicate. ^***P<0.0005, ^**P<0.005

Figure 7

miR-210 inhibition reduces HIF activity in hypoxic A549 cells. A549 cells were transfected with either pre-miR-Neg, pre-miR-210, a control anti-miR-159s LNA or anti-miR-210 in normoxia or hypoxia (1% O₂) and incubated for 48–72 h. (a) Protein levels of HIF-1α and of the loading control α-tubulin. (b) A549 cells were co-transfected with an HRE luciferase reporter and HIF activity was assessed using the dual luciferase reporter assay system at 48 h following transfection. Determinations were done in triplicate and the experiment was repeated three times. (c) The extracellular lactate concentration of transfected cells incubated in normoxia or hypoxia 1% O₂ for 48 h was normalized to cell protein for each condition. Mean±S.E.M. is representative of three independent experiments carried out in triplicate. (d) Proposed model for miR-210, SDHD and HIF-1α inter-regulation. HIF-1α upregulates miR-210, which in turn downregulates SDHD, leading to complex II dysfunction. High succinate levels resulting from loss of complex II could inhibit the PHD activity giving HIF-1α stabilization. ^**P<0.005, ^*P<0.05