Hypoxia-induced changes in expression levels of miR-210, -183 and -744 in human lung fibroblasts

Aims: Micro-RNA (miRNA) profiles of human pulmonary arterial fibroblasts (hPAFb) were measured in order to reveal hypoxia-induced changes in miRNA profiles. Pathway analysis based on miRNA target prediction was compared with pathway analysis based on mRNA microarrays. Methods: Fibroblasts from pulmonary arteries of a healthy human lung were cultured and kept under normoxic and hypoxic (FiO2=0.1) conditions for 24h. Total RNA was isolated using a phenol-/guanidine-based method. Success of the hypoxic treatment was confirmed by measuring the expression of the PGK1 gene by quantitative real-time PCR (qRT-PCR). The miRNA was labelled by T4-Ligase-mediated linking of Cy-Dye-conjugated oligoribonucleotides. Labelled miRNAs were hybridized to miChip microarrays (EMBL, miRBase V11.0). Validation of miRNA expression levels was done with qRT-PCR. miRNA target prediction and KEGG pathway analysis were performed with newly developed target-weighting-algorithms. Results were compared with KEGG pathway analysis based on Agilent Whole Genome microarrays. Results: miR-210, -744 and -296–5p (up) and miR-183 (down) were found regulated in response to hypoxia in microarrays successfully validated by qRT-PCR. Pathway analysis based on the miRNA target scoring algorithm resulted in 31 candidate pathways potentially perturbed by hypoxia-dependet regulation of miRNAs (based on 5% FDR). Most significant significantly enriched pathways are involved in cytoskeletal devopment (Focal Adhesion, ECM-Receptor interaction, Regulation of actin cytoskeleton, Adherens junction, Axon guidance), signalling (Wnt-signalling, mTOR-signalling, TGFβ-signalling) and cell division (Pathways in cancer). 60% of the predicted pathways were confirmed by mRNA expression profiles. Conclusions: Since the HIF-1α- and -2α-induced mir-210 is a major hypoxia-regulated miRNA, its regulation confirms the validity of our results. It is involved in cell cycle arrest, stem cell survival, mitochondrial metabolism, DNA-repair, angiogenesis and cell differentiation. However, none of the other regulated miRNAs has yet been linked to hypoxia in hPAFb. miR-183 is connected to cellular stress and also differenzially expressed in several cancers. miR-744 is a post-transcriptional regulator of TGFβ-1. The pathway prediction based on weighting algorithms seems to be a viable approach as its results are comparable to results based on real mRNA expression data. The reliability of results will likely improve with better target gene annotations as they will be available in future.