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Integrated analysis of microRNA and mRNA expression and association with HIF binding reveals the complexity of microRNA expression regulation under hypoxia.

Camps C, Saini HK, Mole DR, Choudhry H, Reczko M, Guerra-Assunção JA, Tian YM, Buffa FM, Harris AL, Hatzigeorgiou AG, Enright AJ, Ragoussis J - Mol. Cancer (2014)

Bottom Line: SiRNA against HIF-1α and HIF-2α were performed as previously published.Moreover the expression of hsa-miR-27a-3p and hsa-miR-24-3p was found positively associated to a hypoxia gene signature in breast cancer.Gene expression analysis showed no full coordination between pri-miRNA and microRNA expression, pointing towards additional levels of regulation.

View Article: PubMed Central - HTML - PubMed

Affiliation: The Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford, United Kingdom. ccamps@well.ox.ac.uk.

ABSTRACT

Background: In mammalians, HIF is a master regulator of hypoxia gene expression through direct binding to DNA, while its role in microRNA expression regulation, critical in the hypoxia response, is not elucidated genome wide. Our aim is to investigate in depth the regulation of microRNA expression by hypoxia in the breast cancer cell line MCF-7, establish the relationship between microRNA expression and HIF binding sites, pri-miRNA transcription and microRNA processing gene expression.

Methods: MCF-7 cells were incubated at 1% Oxygen for 16, 32 and 48 h. SiRNA against HIF-1α and HIF-2α were performed as previously published. MicroRNA and mRNA expression were assessed using microRNA microarrays, small RNA sequencing, gene expression microarrays and Real time PCR. The Kraken pipeline was applied for microRNA-seq analysis along with Bioconductor packages. Microarray data was analysed using Limma (Bioconductor), ChIP-seq data were analysed using Gene Set Enrichment Analysis and multiple testing correction applied in all analyses.

Results: Hypoxia time course microRNA sequencing data analysis identified 41 microRNAs significantly up- and 28 down-regulated, including hsa-miR-4521, hsa-miR-145-3p and hsa-miR-222-5p reported in conjunction with hypoxia for the first time. Integration of HIF-1α and HIF-2α ChIP-seq data with expression data showed overall association between binding sites and microRNA up-regulation, with hsa-miR-210-3p and microRNAs of miR-27a/23a/24-2 and miR-30b/30d clusters as predominant examples. Moreover the expression of hsa-miR-27a-3p and hsa-miR-24-3p was found positively associated to a hypoxia gene signature in breast cancer. Gene expression analysis showed no full coordination between pri-miRNA and microRNA expression, pointing towards additional levels of regulation. Several transcripts involved in microRNA processing were found regulated by hypoxia, of which DICER (down-regulated) and AGO4 (up-regulated) were HIF dependent. DICER expression was found inversely correlated to hypoxia in breast cancer.

Conclusions: Integrated analysis of microRNA, mRNA and ChIP-seq data in a model cell line supports the hypothesis that microRNA expression under hypoxia is regulated at transcriptional and post-transcriptional level, with the presence of HIF binding sites at microRNA genomic loci associated with up-regulation. The identification of hypoxia and HIF regulated microRNAs relevant for breast cancer is important for our understanding of disease development and design of therapeutic interventions.

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Hypoxia regulates genes involved in microRNA processing pathway in a HIF-dependent manner. QPCR was performed to monitor the expression of genes involved in the microRNA processing pathway. Hypoxia MCF-7 time course and 2 sets of HIF-1α and HIF-2α siRNA in MCF-7 were analysed. Fold-changes (in linear scale) obtained for each gene at each time point relative to corresponding control are represented in boxplots (*significant fold-change compared to normoxia after ANOVA followed by pairwise t-test, adj.p-val ≤ 0.05; † adj.p-val ≤0.08).
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Figure 6: Hypoxia regulates genes involved in microRNA processing pathway in a HIF-dependent manner. QPCR was performed to monitor the expression of genes involved in the microRNA processing pathway. Hypoxia MCF-7 time course and 2 sets of HIF-1α and HIF-2α siRNA in MCF-7 were analysed. Fold-changes (in linear scale) obtained for each gene at each time point relative to corresponding control are represented in boxplots (*significant fold-change compared to normoxia after ANOVA followed by pairwise t-test, adj.p-val ≤ 0.05; † adj.p-val ≤0.08).

Mentions: Overall there is a certain degree of coordination between host gene and microRNA expression under hypoxia but also discrepancy. This suggests that other factors may be involved in the changes observed in mature microRNA expression under hypoxia. We used the gene expression profiles to specifically monitor any change in the expression of genes coding for proteins involved in the microRNA maturation pathway in hypoxic MCF-7 cells. Indeed, significant changes (see Additional file 1: Table S7, adj.p-val < 0.05) were detected in the expression of several genes, including DDX5 (encoding P68 which forms part of the protein complex lead by DROSHA), XPO5 and RAN (encoding EXPORTIN5 and RAN, respectively, both involved in microRNA transport from the nucleus to the cytoplasm), DICER and EIF2C2 and EIF2C4 (encoding AGO2 and AGO4, respectively, both associated to RISC complex). We also found that some of these changes were HIF-1α and HIF-2α dependent based on previous data, in which gene expression regulations dependent on these transcription factors were studied through microarray profiles after short interfering (si) RNA-based suppression of HIF subunits [26]. In order to validate these results, we performed qPCR using the MCF-7 hypoxia time course, and two HIF-1α and HIF-2α siRNA sample sets (Figure 6). Indeed, we could confirm a significant down-regulation of DDX5, XPO5, RAN, EIF2C2 and DICER consistently across the hypoxia time course in MCF-7 cells, as well as a significant up-regulation of EIF2C4 (Figure 6). We also confirmed by qPCR a significant up-regulation of both genes DICER and EIF2C2 when HIF-1α and HIF-2α are silenced in MCF-7 compared to the control, in both independent sample sets (Figure 6). Concerning EIF2C4, its expression was found significantly down-regulated upon HIF-1α silencing (Figure 6). Therefore the down-regulation of DICER and EIF2C2 under hypoxia is dependent on both HIF-1α and HIF-2α, whereas the up-regulation of EIF2C4 seems to be only dependent on HIF-1α. Changes observed in DDX5, XPO5 and RAN in hypoxic MCF-7 seem to be independent of HIF-1α or HIF-2α, as their expression did not change in the HIF siRNA experiments (Figure 6).


Integrated analysis of microRNA and mRNA expression and association with HIF binding reveals the complexity of microRNA expression regulation under hypoxia.

Camps C, Saini HK, Mole DR, Choudhry H, Reczko M, Guerra-Assunção JA, Tian YM, Buffa FM, Harris AL, Hatzigeorgiou AG, Enright AJ, Ragoussis J - Mol. Cancer (2014)

Hypoxia regulates genes involved in microRNA processing pathway in a HIF-dependent manner. QPCR was performed to monitor the expression of genes involved in the microRNA processing pathway. Hypoxia MCF-7 time course and 2 sets of HIF-1α and HIF-2α siRNA in MCF-7 were analysed. Fold-changes (in linear scale) obtained for each gene at each time point relative to corresponding control are represented in boxplots (*significant fold-change compared to normoxia after ANOVA followed by pairwise t-test, adj.p-val ≤ 0.05; † adj.p-val ≤0.08).
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Related In: Results  -  Collection

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Figure 6: Hypoxia regulates genes involved in microRNA processing pathway in a HIF-dependent manner. QPCR was performed to monitor the expression of genes involved in the microRNA processing pathway. Hypoxia MCF-7 time course and 2 sets of HIF-1α and HIF-2α siRNA in MCF-7 were analysed. Fold-changes (in linear scale) obtained for each gene at each time point relative to corresponding control are represented in boxplots (*significant fold-change compared to normoxia after ANOVA followed by pairwise t-test, adj.p-val ≤ 0.05; † adj.p-val ≤0.08).
Mentions: Overall there is a certain degree of coordination between host gene and microRNA expression under hypoxia but also discrepancy. This suggests that other factors may be involved in the changes observed in mature microRNA expression under hypoxia. We used the gene expression profiles to specifically monitor any change in the expression of genes coding for proteins involved in the microRNA maturation pathway in hypoxic MCF-7 cells. Indeed, significant changes (see Additional file 1: Table S7, adj.p-val < 0.05) were detected in the expression of several genes, including DDX5 (encoding P68 which forms part of the protein complex lead by DROSHA), XPO5 and RAN (encoding EXPORTIN5 and RAN, respectively, both involved in microRNA transport from the nucleus to the cytoplasm), DICER and EIF2C2 and EIF2C4 (encoding AGO2 and AGO4, respectively, both associated to RISC complex). We also found that some of these changes were HIF-1α and HIF-2α dependent based on previous data, in which gene expression regulations dependent on these transcription factors were studied through microarray profiles after short interfering (si) RNA-based suppression of HIF subunits [26]. In order to validate these results, we performed qPCR using the MCF-7 hypoxia time course, and two HIF-1α and HIF-2α siRNA sample sets (Figure 6). Indeed, we could confirm a significant down-regulation of DDX5, XPO5, RAN, EIF2C2 and DICER consistently across the hypoxia time course in MCF-7 cells, as well as a significant up-regulation of EIF2C4 (Figure 6). We also confirmed by qPCR a significant up-regulation of both genes DICER and EIF2C2 when HIF-1α and HIF-2α are silenced in MCF-7 compared to the control, in both independent sample sets (Figure 6). Concerning EIF2C4, its expression was found significantly down-regulated upon HIF-1α silencing (Figure 6). Therefore the down-regulation of DICER and EIF2C2 under hypoxia is dependent on both HIF-1α and HIF-2α, whereas the up-regulation of EIF2C4 seems to be only dependent on HIF-1α. Changes observed in DDX5, XPO5 and RAN in hypoxic MCF-7 seem to be independent of HIF-1α or HIF-2α, as their expression did not change in the HIF siRNA experiments (Figure 6).

Bottom Line: SiRNA against HIF-1α and HIF-2α were performed as previously published.Moreover the expression of hsa-miR-27a-3p and hsa-miR-24-3p was found positively associated to a hypoxia gene signature in breast cancer.Gene expression analysis showed no full coordination between pri-miRNA and microRNA expression, pointing towards additional levels of regulation.

View Article: PubMed Central - HTML - PubMed

Affiliation: The Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford, United Kingdom. ccamps@well.ox.ac.uk.

ABSTRACT

Background: In mammalians, HIF is a master regulator of hypoxia gene expression through direct binding to DNA, while its role in microRNA expression regulation, critical in the hypoxia response, is not elucidated genome wide. Our aim is to investigate in depth the regulation of microRNA expression by hypoxia in the breast cancer cell line MCF-7, establish the relationship between microRNA expression and HIF binding sites, pri-miRNA transcription and microRNA processing gene expression.

Methods: MCF-7 cells were incubated at 1% Oxygen for 16, 32 and 48 h. SiRNA against HIF-1α and HIF-2α were performed as previously published. MicroRNA and mRNA expression were assessed using microRNA microarrays, small RNA sequencing, gene expression microarrays and Real time PCR. The Kraken pipeline was applied for microRNA-seq analysis along with Bioconductor packages. Microarray data was analysed using Limma (Bioconductor), ChIP-seq data were analysed using Gene Set Enrichment Analysis and multiple testing correction applied in all analyses.

Results: Hypoxia time course microRNA sequencing data analysis identified 41 microRNAs significantly up- and 28 down-regulated, including hsa-miR-4521, hsa-miR-145-3p and hsa-miR-222-5p reported in conjunction with hypoxia for the first time. Integration of HIF-1α and HIF-2α ChIP-seq data with expression data showed overall association between binding sites and microRNA up-regulation, with hsa-miR-210-3p and microRNAs of miR-27a/23a/24-2 and miR-30b/30d clusters as predominant examples. Moreover the expression of hsa-miR-27a-3p and hsa-miR-24-3p was found positively associated to a hypoxia gene signature in breast cancer. Gene expression analysis showed no full coordination between pri-miRNA and microRNA expression, pointing towards additional levels of regulation. Several transcripts involved in microRNA processing were found regulated by hypoxia, of which DICER (down-regulated) and AGO4 (up-regulated) were HIF dependent. DICER expression was found inversely correlated to hypoxia in breast cancer.

Conclusions: Integrated analysis of microRNA, mRNA and ChIP-seq data in a model cell line supports the hypothesis that microRNA expression under hypoxia is regulated at transcriptional and post-transcriptional level, with the presence of HIF binding sites at microRNA genomic loci associated with up-regulation. The identification of hypoxia and HIF regulated microRNAs relevant for breast cancer is important for our understanding of disease development and design of therapeutic interventions.

Show MeSH
Related in: MedlinePlus