MIR-210 Induces Angiogenesis and Endothelin-2 Expression In Human Granulosa Cells Under Normoxic
and Hypoxic Conditions

Hypoxia is known to induce endothelin-2 (EDN-2) and vascular endothelial growth factor (VEGF) expression in the preovulatory granulosa cells. miR-210 is up regulated in response to hypoxia-inducible factors (HIF). We studied the effect of miR-210 on HIF-1α protein and its inducible genes in normoxic and hypoxic conditions in human granulosa cell line. Methods: miR 210 was analyzed in a tissue culture model of SVOG cells. Cells were transfected with either synthetic miR 210 molecules or specific inhibitor of miR 210. Cells were cultured in normoxic and hypoxic conditions for 24 hours post transfection. EDN-2 and VEGF mRNA expression levels were assayed using real time PCR with the specific primers. HIF-1α protein level was measured using immunoblotting. Results: The induced elevation of miR-210 expression led to increased elevation of HIF 1α levels under hypoxic conditions. The EDN-2 and VEGF expression was 2 fold higher in transfected cells under normoxic conditions. After 24h of induced hypoxia the transfected cells significantly increased their expression of EDN-2 and VEGF mRNA. Cells transfected with miR-210 inhibitor demonstrated reduced expression levels of EDN-2 and VEGF in normoxic as well as in hypoxic conditions. Conclusions: miR-210 increases the expression of HIF-1α protein under hypoxic conditions in human granulosa cells and also increases the expression of EDN-2 and VEGF. This occurs also without the presence of hypoxia. Antagonizing miR-210 effect abolishes these normoxic and hypoxic effects. Our results suggest an independent effect of miR 210 on angiogenesis and on the endothelin system in the active human corpus luteum.