Expression of sFLT-1 and JMJD6 in Cultured Human Trophoblasts

Abstract

Placenta Growth Factor (PlGF) plays an important role in the formation and growth of the placenta. This protein binds two receptors, a membrane bound Flt-1 (mFlt-1) and a soluble Flt-1 (sFlt-1), the latter of which lacks intracellular signaling. These receptors are splice variants of each other and differ in their intracellular region. Although we have shown that hypoxia increases the expression of sFlt -1 in trophoblast, the mechanism for this is not known. Silencing Jumoji domain-containing protein 6 (Jmjd6) expression in endothelial cells increases soluble Flt-1 expression and hypoxia lowers the expression of Jmjd6 in endothelial cells. However, it is not known if Jmjd6 is found in trophoblasts or if it functions in determining the splicing of Flt-1 in these cells. This study sought to optimize an assay to assess Jmjd6 expression in trophoblast, how its expression is affected by hypoxia, and how this correlates to sFlt-1 expression. Trophoblast were isolated from three different normal placentae and cultured for 24 hours under two conditions: normoxia (21% oxygen) and hypoxia (1-2% oxygen). The RNA was harvested, reversed transcribed into cDNA and sFlt-1/Jmjd6 expression was analyzed in duplicate using real-time PCR. The fold changes in sFlt-1 between the normoxic and hypoxic groups were 1.98, 0.75, and 0.88, for the samples. We optimized the PCR of Jmjd6 by changing annealing temperature and concentrations of primers and cDNA. The fold changes in Jmjd6 are still being determined. In conclusion we determined Jmjd6 is expressed in trophoblast but its influence on sFlt-1 expression is unknown. 32