Abstract 036: Regulation of sFlt-1 Splicing by U2AF and JMJD

Abstract

Preeclampsia (PE) is a gestational disorder defined by new-onset hypertension and maternal endothelial dysfunction. Though its origins are unclear, placental ischemia is thought to be the central cause. In response, the placenta releases pathogenic factors, such as the anti-angiogenic soluble form of the VEGF receptor Flt-1 (sFlt-1), into the maternal circulation. sFlt-1 is a soluble splice variant of the VEGF receptor Flt-1, acting as a VEGF sink in the circulation. The regulation of sFlt-1 splicing is not completely understood, but it is proposed that the splicing factor U2AF65 promotes splicing, while JMJD6 opposes it by degrading U2AF65. Here, we test the hypothesis that decreased expression of U2AF65 will decrease sFlt-1 release, while decreased JMJD6 will increase sFlt-1 release. Using immortalized placental trophoblasts (BeWo), siRNA was used to knock down the expression of U2AF65, confirmed by RT-qPCR (1.014 + 0.12 vs 0.379 + 0.131; p<0.05). After transfection, cells were incubated at 8% and 1% oxygen for 24 hours, mimicking the conditions found in the healthy and ischemic placenta respectively. As hypothesized, U2AF65 knockdown caused a 52% decrease in released sFlt-1 compared to controls in hypoxia (56.8 + 9.4 pg/mL vs 118.9 + 9.2 pg/mL; p<0.05). siRNA successfully reduced JMDJ6 mRNA (1.008 + 0.09 vs 0.3698 + 0.04; p<0.05) and demonstrated undetectable protein levels. Unexpectedly, this resulted in a 33.8% decrease (342.7 + 35.8 pg/mL vs 517.8 + 15.4 pg/mL; p<0.005) in the amount of sFlt-1 release from cells in hypoxia and a 49% decrease (246.2 + 23.6 pg/mL vs 482.8 + 39.4 pg/mL; p<0.001) in normoxia compared to their respective controls. We also measured both U2AF65 and JMJD6 in the placentas of control rats and those that had chronic placental ischemia due to the Reduced Uterine Perfusion Pressure (RUPP) procedure. RUPP placentas showed a 61.6% increase in U2AF65 compared to controls (161.6 + 11.6 AU vs 100 + 9.8 AU; p<0.005) and a 121% increase in JMJD6 in RUPP placentas (221.1 + 30.4 AU vs 100 + 40.9 AU; p= 0.0549). Based on these data, we speculate that both JMJD6 and U2AF65 are crucial for the production of sFlt-1 by affecting alternative splicing. Targeting these factors could prove a useful approach for reducing sFlt-1 production in the preeclampsia patient.