MicroRNA response to hypoxic stress in soft tissue sarcoma cells: microRNA mediated regulation of HIF3α

Background: Hypoxia is often encountered in solid tumors and known to contribute to aggressive tumor behavior, radiation- and chemotherapy resistance resulting in a poor prognosis for the cancer patient. MicroRNAs (miRNAs) play a role in the regulation of the tumor cell response to hypoxia, however, not much is known about the involvement of miRNAs in hypoxic signalling pathways in soft tissue sarcomas (STS). Method: A panel of twelve STS cell lines was exposed to atmospheric oxygen concentrations (normoxia) or 1% oxygen (hypoxia) for up to 48 h. Hypoxic conditions were verified and miRNA expression profiles were assessed by LNA (TM) oligonucleotide microarrays and RT-PCR after 24 h. The expression of target genes regulated by hypoxia responsive miRNAs is examined by end-point PCR and validated by luciferase reporter constructs. Results: Exposure of STS cell lines to hypoxic conditions gave rise to upregulation of Hypoxia Inducible Factor (HIF) 1 alpha protein levels and increased mRNA expression of HIF1 target genes CA9 and VEGFA. Deregulation of miRNA expression after 24 h of hypoxia was observed. The most differentially expressed miRNAs ( p < 0.001) in response to hypoxia were miR-185-3p, miR-485-5p, miR-216a-5p (upregulated) and miR-625-5p (downregulated). The well-known hypoxia responsive miR-210-3p could not be reliably detected by the microarray platform most likely for technical reasons, however, its upregulation upon hypoxic stress was apparent by qPCR. Target prediction algorithms identified 11 potential binding sites for miR-485-5p and a single putative miR-210-3p binding site in the 3'UTR of HIF3 alpha, the least studied member of the HIF family. We showed that HIF3 alpha transcripts, expressing a 3'UTR containing the miR-485-5p and miR-210-3p target sites, are expressed in all sarcoma cell lines and upregulated upon hypoxia. Additionally, luciferase reporter constructs containing the 3'UTR of HIF3 alpha were used to demonstrate regulation of HIF3 alpha by miR-210-3p and miR-485-5p. Conclusion: Here we provide evidence for the miRNA mediated regulation of HIF3 alpha by hypoxia responsive miRNAs in STS, which may help to tightly regulate and fine-tune the hypoxic response. This provides a better insight into the mechanisms underlying the hypoxic response in STS and may ultimately yield information on novel prognostic and predictive markers or targets for treatment.