The placental renin-angiotensin system, particularly prorenin and the (pro)renin receptor ((P)RR), are highly expressed in the early gestation placenta, when extravillous trophoblast migration and invasion is maximal. If trophoblast invasion and artery remodelling are inadequate, this can lead to shallow placentation and abnormal pregnancy outcomes. Our study aims to investigate the role of the (P)RR in early placental development.
A first trimester extravillous trophoblast cell line, HTR-8/SVneo, was transfected with siRNA targeting (P)RR mRNA. This was used either alone or in combination with an siRNA targeting the angiotensin II type 1 receptor (AT1R). Rates of proliferation and migration after (P)RR and/or AT1R knockdown were monitored in real-time using the xCELLigence Real-Time Cell Analysis system. In order to identify novel downstream pathways affected by knockdown of (P)RR, samples were prepared for proteomics analysis via liquid chromatography-mass spectrometry (LC-MS) using the TMT-10Plex Mass Tag labelling system.
The rate of trophoblast cell proliferation was significantly decreased when treated with (P)RR siRNA alone (P=0.03) or (P)RR siRNA + AT1R siRNA used in combination (P<0.006). AT1R siRNA alone had no effect on cell proliferation. Similarly, the rate of cell migration was significantly decreased in cells treated with (P)RR siRNA alone (P=0.0001) and (P)RR siRNA + AT1R siRNA used in combination (P<0.007) but not with AT1R siRNA alone. Analysis of mass spectrometry data showed that a number of phosphorylated cytoskeletal proteins were significantly upregulated in HTR-8/SVneo cells when (P)RR was knocked down compared to cells treated with negative control siRNA. These included metastasis suppressor 1 (MTSS1; P<0.03), kinectin (KTN1; P<0.03), and cytopsin (SPECC1;P<0.04).
These preliminary data suggest that proliferation and migration are regulated via (P)RR and not Ang II/AT1R, and that (P)RR regulates pathways involved in cytoskeletal remodelling, which could explain the decrease in HTR-8/SVneo cell proliferation and migration after (P)RR knockdown.