MicroRNA regulates vascular endothelial growth factor expression in chondrosarcoma cells.

backgroundSystemic treatments to prevent or treat chondrosarcoma metastasis are lacking and targeted therapy has yet to be developed. Hypoxia develops in tumors as they grow and hypoxia-related alterations in gene expression underlie some of the traits of cancer. One critical trait is the ability to induce sustained angiogenesis, which is usually related to expression of vascular endothelial growth factor (VEGF). A potential hypoxia-related mechanism resulting in altered gene expression involves microRNA. Little is known about microRNA expression in chondrosarcoma and its potential role in regulation of VEGF expression. QUESTIONS/PURPOSES: Our purposes were (1) to determine if there is hypoxia-regulated microRNA overexpressed in chondrosarcoma; (2) if that contributes to increased VEGF expression; and (3) can VEGF expression be inhibited with a specific antagomir?

methodsMicroRNA expression was analyzed in two primary human chondrosarcomas and articular cartilage using array analysis and a cutoff of a fourfold difference in expression between tumor and normal tissue. The effects of hypoxia and hypoxia-inducible factor-1α (HIF-1α) transfection and silencing with siRNA on expression of candidate microRNAs were analyzed in chondrosarcoma cell line JJ. VEGF expression was measured with quantitative polymerase chain reaction and enzyme-linked immunosorbent assay after specific microRNA transfection and knockdown.

resultsmiR-181a was identified by array analysis and confirmed with quantitative reverse transcription-polymerase chain reaction, which showed that miR-181a was overexpressed in both human chondrosarcomas (33- and 55-fold) and the JJ cell line (sixfold) compared with cartilage and chondrocytes, respectively. In vitro, hypoxia and HIF-1α transfection each further increased miR-181a expression twofold in JJ cells. miR-181a transfection of JJ cells doubled expression of VEGF mRNA and increased secreted VEGF protein by 46% in normoxia, an effect that could be either direct or indirect. Similar enhancement of VEGF expression by miR-181a was found during hypoxia. Transfection with the antagomir anti-miR-181a decreased VEGF protein by 27% in normoxia and 23% in hypoxia.

conclusionsmiR-181a is a hypoxia-regulated microRNA that is overexpressed in chondrosarcoma and enhances VEGF expression, an effect that could be inhibited by anti-miR-181a. CLINICAL RELEVANCE: Systemic treatment options for chondrosarcoma are limited. Antiangiogenic strategies could potentially be effective in limiting tumor progression. One method of inhibiting VEGF expression and associated angiogenesis could be an antagomir-based therapy targeted at miR-181a or other oncogenic microRNAs, although methods of systemic delivery are still under development. The effectiveness of antagomirs also needs to be compared with other antiangiogenic modalities in preclinical models.