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 welldocumented 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 hypoxiainduced microRNAs are also overexpressed in a variety of human tumors.

Genetic instability is a hallmark of cancer; the hypoxic tumor microenvironment has been implicated as a cause of this phenomenon. MicroRNAs (miR) are small nonprotein coding RNAs that can regulate various cellular pathways. We report here that two miRs, miR-210 and miR-373, are up-regulated in a hypoxia-inducible factor-1A-dependent manner in hypoxic cells. Bioinformatics analyses suggested that these miRs could regulate factors implicated in DNA repair pathways. Forced expression of miR-210 was found to suppress the levels of RAD52, which is a key factor in homology-dependent repair (HDR); the forced expression of miR-373 led to a reduction in the nucleotide excision repair (NER) protein, RAD23B, as well as in RAD52. Consistent with these results, both RAD52 and RAD23B were found to be down-regulated in hypoxia, but in both cases, the hypoxia-induced down-regulation could be partially reversed by antisense inhibition of miR-210 and miR-373. Importantly, luciferase reporter assays indicated that miR-210 is capable of interacting with the 3 ¶ untranslated region (UTR) of RAD52 and that miR-373 can act on the 3 ¶ UTR of RAD23B. These results indicate that hypoxia-inducible miR-210 and miR-373 play roles in modulating the expression levels of key proteins involved in the HDR and NER pathways, providing new mechanistic insight into the effect of hypoxia on DNA repair and genetic instability in cancer. [Cancer Res 2009;69(3):1221-9]

miR-210 is a key player of cell response to hypoxia, modulating cell survival, VEGF-driven endothelial cell migration, and the ability of endothelial cells to form capillary-like structures. A crucial step in understanding microRNA (miRNA) function is the identification of their targets. However, only few miR-210 targets have been identified to date. Here, we describe an integrated strategy for large-scale identification of new miR-210 targets by combining transcriptomics and proteomics with bioinformatic approaches. To experimentally validate candidate targets, the RNA-induced silencing complex (RISC) loaded with miR-210 was purified by immunoprecipitation along with its mRNA targets. The complex was significantly enriched in mRNAs of 31 candidate targets, such as BDNF, GPD1L, ISCU, NCAM, and the non-coding RNA Xist. A subset of the newly identified targets was further confirmed by 3-untranslated region (UTR) reporter assays, and hypoxia induced down-modulation of their expression was rescued blocking miR-210, providing support for the approach validity. In the case of 9 targets, such as PTPN1 and P4HB, miR-210 seed-pairing sequences localized in the coding sequence or in the 5-UTR, in line with recent data extending miRNA targeting beyond the "classic" 3-UTR recognition. Finally, Gene Ontology analysis of the targets highlights known miR-210 impact on cell cycle regulation and differentiation, and predicts a new role of this miRNA in RNA processing, DNA binding, development, membrane trafficking, and amino acid catabolism. Given the complexity of miRNA actions, we view such a multiprong approach as useful to adequately describe the multiple pathways regulated by miR-210 during physiopathological processes.miRNAs are 21-23-nucleotide non-protein coding RNA molecules that regulate the stability and/or the translational efficiency of target messenger RNAs (1-3). Mature miRNAs are loaded into the RNA-induced silencing complex (RISC) 3 and mediate the translational inhibition of target mRNA, albeit a few opposing examples have been described as well (4 -6). The rules that guide miRNA-mRNA interaction are very complex and still under investigation. However, the current paradigm states that a Watson-Crick pairing between the mRNA and the 5Ј-region of the miRNA centered on nucleotides 2-7, termed "seed sequence," is required for miRNA-mediated inhibition (7). RISC-miRNA complexes can move the mRNAs they bind to the P-bodies, which are specialized cytoplasmic compartments where translational repression and mRNA turnover is thought to occur (8). Because P-bodies contain many enzymes involved in mRNA exonucleolitic degradation, miRNAs may also have a secondary quantitative inhibitory effect on mRNAs. A role for miRNAs in mRNA destabilization is also suggested by studies reporting robust correlations between the levels of miRNAs and the message of multiple predicted or validated targets (9 -11).miR-210 is currently regarded as "master miRNA" of hypoxic response, because it was found up-regulated by hypoxia in all the ce...

microRNAs participate in a wide variety of physiological and pathological cellular processes. Recent studies have established a link between a specific group of microRNAs and hypoxia, a key feature of the neoplastic microenvironment. A significant proportion of the hypoxia-regulated microRNAs (HRMs) are also overexpressed in human cancers, suggesting a role in tumorigenesis. Preliminary evidence suggests that they could affect important processes such as apoptosis, proliferation and angiogenesis. Several HRMs exhibit induction in response to HIF activation, thus extending its repertoire of targets beyond translated genes. In the present review, we discuss the emerging roles of HRMs in oxygen deprivation in cancer context.

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MicroRNAs (miRs) are short non-coding transcripts involved in a wide variety of cellular processes. Several recent studies have established a link between hypoxia, a well-documented component of the tumour microenvironment, and specific miRs. One member of this class, miR-210, was identified as hypoxia inducible in all the cell types tested, and is overexpressed in most cancer types. Its hypoxic induction is dependent on a functional hypoxia-inducible factor (HIF), thus extending the transcriptional repertoire of the latter beyond ‘classic’ genes. From a clinical standpoint, miR-210 overexpression has been associated with adverse prognosis in breast tumours and been detected in serum of lymphoma patients and could serve as a tool to define hypoxic malignancies. We discuss the role of miR-210 and its emerging targets, as well as possible future directions for clinical applications in oncology and ischaemic disorders.

BackgroundMicroRNAs are a class of small non-coding RNAs that regulate mRNA expression at the post - transcriptional level and thereby many fundamental biological processes. A number of methods, such as multiplex polymerase chain reaction, microarrays have been developed for profiling levels of known miRNAs. These methods lack the ability to identify novel miRNAs and accurately determine expression at a range of concentrations. Deep or massively parallel sequencing methods are providing suitable platforms for genome wide transcriptome analysis and have the ability to identify novel transcripts.ResultsThe results of analysis of small RNA sequences obtained by Solexa technology of normal peripheral blood mononuclear cells, tumor cell lines K562 and HL60 are presented. In general K562 cells displayed overall low level of miRNA population and also low levels of DICER. Some of the highly expressed miRNAs in the leukocytes include several members of the let-7 family, miR-21, 103, 185, 191 and 320a. Comparison of the miRNA profiles of normal versus K562 or HL60 cells revealed a specific set of differentially expressed molecules. Correlation of the miRNA with that of mRNA expression profiles, obtained by microarray, revealed a set of target genes showing inverse correlation with miRNA levels. Relative expression levels of individual miRNAs belonging to a cluster were found to be highly variable. Our computational pipeline also predicted a number of novel miRNAs. Some of the predictions were validated by Real-time RT-PCR and or RNase protection assay. Organization of some of the novel miRNAs in human genome suggests that these may also be part of existing clusters or form new clusters.ConclusionsWe conclude that about 904 miRNAs are expressed in human leukocytes. Out of these 370 are novel miRNAs. We have identified miRNAs that are differentially regulated in normal PBMC with respect to cancer cells, K562 and HL60. Our results suggest that post - transcriptional processes may play a significant role in regulating levels of miRNAs in tumor cells. The study also provides a customized automated computation pipeline for miRNA profiling and identification of novel miRNAs; even those that are missed out by other existing pipelines. The Computational Pipeline is available at the website: http://mirna.jnu.ac.in/deep_sequencing/deep_sequencing.html

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