Hypoxia induces changes in miRNA expression. (A) Unsupervised hierarchical clustering using miRNA expression data (407 miRNAs) of hypoxic (H) and normoxic (N) cell line samples does not discriminate the hypoxic and normoxic samples. The hypoxic samples cluster together with their normoxic counterparts. (B) The most significant differentially expressed miRNAs (two-sample t-test, p < 0.05) between cell lines that were cultured under normoxic and hypoxic conditions are used for a supervised hierarchical clustering. The expression of these 32 miRNAs can distinguish hypoxic from normoxic sarcoma cell line samples. (C) The four most significant (P < 0.001) differentially expressed miRNAs (miR-485-5p, miR-216a-5p, miR-185-3p and miR-625-5p) discriminate sarcoma cell lines that were cultured under normoxic conditions from those cultured under hypoxic conditions in a supervised hierarchical clustering. (D) MiR-210-3p is upregulated during hypoxia. RT-PCR was used to determine miR-210-3p levels in normoxic (black bars) and hypoxic (grey bars) samples from five representative sarcoma cell lines. Expression is normalized against RNU43 expression. Bars indicate average expression fold change ± S.D. (n = 2) of miR-210-3p compared to the expression at 0 h. Statistical significance between expression in normoxic and hypoxic samples at a time point were determined by two-sample t-tests: * = p < 0.05, ** = p < 0.005, *** = p < 0.0005. The different cell lines are indicated by a color code that identifies their tumor of origin: with synovial sarcoma (yellow); Ewing sarcoma (green); rhabdomyosarcoma (purple); leiomyosarcoma (blue); liposarcoma (orange) and fibrosarcoma (red).