Delivery of AP-2α siRNA into cultured bovine trophoblast cells by electroporation repressed key placenta-specific gene expression

Abstract

Binucleate trophoblast giant cells (BNC) characteristically appear early in gestation in the bovine placenta. They secret pivotal hormones and cytokines for feto-maternal communication, for example, expression of placental lactogens (CSH1), prolactin-related protein 1 (PRP1) and pregnancy-associated glycoprotein 1 (PAG1) are necessary for pregnancy establishment in bovine. These genes transcription are regulated in a temporal and spatial manner, however, molecular mechanisms by which these gene transcriptions are regulated in this manner have not been firmly elucidated. In this study, a cell culture model for bovine trophoblast cells was initially established, small interfering RNA duplexes against Activator Protein-2α (TFAP2A) was transfected into the cells by electroporation, and transcripts of CSH1, PRP1 and PAG1 were measured by qPCR. The results showed that trophoblast giant cells were confluent for 90% after cultured for 10 days, and BNC constituted of a population of more than 45% of the total cells. Using a fluorescein-labeled non-silencing siRNA duplex, an electroporation protocol yielding routinely >93% positive cells could be established, and siRNA duplex transfection demonstrated an efficient knockdown of cellular AP-2α mRNA level by 72.30 ± 3.28% in electroporated cells. Finally, CSH1, PRP1 and PAG1 genes expression were effectively down-regulated by 65.45 ± 6.38% (P<0.01), 40.73±11.72% (P<0.01) and 11.59 ± 1.88% (P<0.05), respectively. It was therefore suggested that electroporating siRNA into bovine trophoblast cells could be an efficient method to manipulate BNC function and to study the regulation mechanism of specific gene transcription without the use of chemical transfection reagents. It was suggested that AP-2α could be at least involved in the regulation of expression CSH1 and PRP1 transcripts.