The Effect Of AP-2α SiRNA Transfection Into Cultured Bovine Binucleate Trophoblast Giant Cells By Electroporation On Expression Of Key Placenta-specific Genes

To study the regulation of activator protein-2a (AP-2a) on the transcripts of bovine binucleate trophoblast giant cells-specific genes including Cshl, PRP-Ⅰand PAG1, BNC were cultured and identified by Hoechst33342 stain and immunocytochemistry to establish a cell culture model for in vitro experiment. Small interfering RNA-mediated AP-2α(AP-2αsiRNA) was electroporated into the cultured cells. Transfection efficiency was analyzed by flow cytometer (FCM) and fluorescence microscope, cells viability was evaluated trypan blue staining after inducing with negative control FAM after culturing for 4h. The optimal electroporation condition and interference site were screened by measuring endogenous TFAP2A with RT-PCR for subsequent experiments. Transcripts of Cshl, PRP-Ⅰand PAG1, the encoding genes of placental lactogens (PL), prolactin-related protein-Ⅰ(PRP-Ⅰ) and pregnancy-associated glycoproteinl(PAG1), respectively, were measured by real-time qPCR.This study could be divided into two parts:1. Cell culture and identificationOne healthy pregnant uteri of Chinese Holstein cow (45-60 days of pregnancy) was collected from a slaughterhouse and the fetal cotyledonary components were manually separated from the maternal caruncle under aseptic condition. The harboured binucleate trophoblast giant cells were seeded on collagenⅠ-coated dishes and cultured in DMEM contained with trophoblast growth supplement and 15% fetal calf serum at 37.5℃with 5% CO2 atmosphere and 100% relative humidity. Cells were stained by Hoechst 33342 and trypan blue. The results showed that cell viability was more than 90%, BNC proportion reached 30% by method of Percoll isolation. BNC presented as epithelial-like shape and maintained an oval flattened shape with the cytoplasm contained many granules were observed, specifically, they regularly displayed two large nuclei. Immunocytochemistry showed that the cultured BNC were positive for cytokeratin and negative for vimentin and positive reactions were detected as red fluorescence. Therefore, it was concluded that a faster and easier proposal for culturing relatively higher proportion of BNC was established.2. Delivery of AP-2asiRNA into Cultured Bovine Trophoblast Cells by ElectroporationNegative control, negative control FAM and three siRNA duplexes, TFAP2A-siRNAs targeting against nucleotides sequence 535,824 and 981 of bovine AP-2a were synthesized. Cells were harvested after culturing for 10 days, siRNA duplexes were transferred to cells via electroporation. Transfection efficiency and cell viability were respectively analyzed by flow cytometer (FCM) and trypan blue staining after inducing with negative control FAM and culturing for 4h. Transcripts of Cshl, PRP-Ⅰand PAG1, the encoding genes of placental lactogens (PL), prolactin-related protein-1 (PRP-Ⅰ) and pregnancy-associated glycoproteinl(PAG1), respectively, were measured by real-time qPCR. Using a fluorescein-labeled non-silencing siRNA duplex, an electroporation protocol yielding routinely>93% positive cells could be established, and siRNA targeting against AP-2a demonstrated an efficient knockdown of cellular mRNA level by 72.30±3.28%(p＜0.01) in electroporated cells. Finally, interference of AP-2a was effective in down-regulation of Cshl, PRP-I and PAG1 gene expression by 65.±6.38%,40.73±11.72%(P<0.01) and 11.59±1.88%(P>0.05), respectively. It was therefore suggested that siRNA electroporation into bovine BNC 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, and AP-2a could be at least involved in regulation of expression Cshl and PRP-Ⅰtranscripts by directly binding to the sites on the upstream of the genes, or by binding with the other family members of AP-2 or cofactors.