Effects of BMPR-IB Gene Silencing by Small Interfering RNA on Apoptosis of Porcine Pollicular Granulosa Cells and Ecpression of BMP Pathy-Way-Ralted Genes
|School||Nanjing Agricultural College|
|Course||Animal Genetic Breeding and Reproduction|
|Keywords||Procine granulosa cells BMPR-IB RNA interference Cell apoptosis|
BMPR-IB, which belongs to type I of BMP receptor family, presents in many types of cells to regulate their growth and differentiation. On sheep, base mutation of BMPR-IB gene leads to changes of amino acid, making part of BMPR-IB gene inactivated and the mechanism of the BMP signal transmission changed, thereby results in insensitive of ovarian granulosa cells(GC) to the inhibition of ligand, ultimately increases the rate of ovulation and fecundity of sheep. Recent researches found irregular estrous cycle and pseudocysis caused by granulosa cells reduction and aggregation on BMPR-IB-deficiency mouse.In this study, effects of BMPR-IB gene silencing on porcine follicular granulosa cells (pGC) were investigated by real time RT-PCR and flow cytometry (FCM);BMPR-IB-siRNA was used to repress the BMP signaling transduction, based on the gene silencing, cells were treated with FSH, and the effects of BMPR-IB gene silencing on FSH-induced GCs were investigated. Cells were treated with BMP-6, an activator of BMP signaling pathway, then the expression of receptors and Smads of downstream were detected by real time RT-PCR.1 BMPR-IB-siRNA transfection of procine Granulosa Cells and valuation of transfection efficiencyAccording to the cDNA sequence of porcine BMPR-IB gene, small interference RNA was synthesized, then BMPR-IB-siRNA were transfected into pGC using liposome. Transfection efficiency was 63.22% by FCM with siRNA concentration of 100nmoL; BMPR-IB mRNA expression level was assayed by real-time RT-PCR. The results showed that the most effective BMPR-IB-siRNA inhibittion at 36h, and the expression level in siRNA group decreased by 65% compared with the control. Apoptpsis rate was detected by FCM, the result showed that it was significanctly increased (16.86% to 10.55%), and the expression of Bcl-2, which was anti-apoptosis gene was significantly declined. The result suggested that the BMPR-IB gene silencing can aggravate apoptosis of pGC. 2 Effects of BMPR-IB gene silencing on apoptosis of FSH-induced porcine follicular granulosa cellsRNAi technology was performed for BMPR-IB gene silencing and FSH was supplemented in the medium of GCs to investigate the effects of BMPR-IB gene silencing on apoptosis of FSH-induced porcine follicular granulosa cells. The apoptosis rate of GCs and the expression of apoptosis-related genes and steroid synthesis-related genes were detected by FCM and real time RT-PCR respectively. The results showed that apoptosis of cells were inhibited by FSH, apoptosis rate were not significantly decreased by FSH based on BMPR-IB gene silencing, and the expression of Bax and Bcl-2, which were original apoptotic gene and anti-apoptosis gene respectively were not significantly changed; FSH can increase the expression of FSHR, but the expression of FSHR were inhibited by the lack of BMPR-IB gene even with the existence of FSH; in addition, the expression of CYP19al and CYP11al gene was increased by FSH, but inhibited by the absence of BMPR-IB gene.3 Expression of BMP pathway-realted genes based on BMP6 after BMPR-IB gene silencingTo study the effects of BMPR-IB-siRNA transfection of pGCs on BMP signal pathway, BMP6 was supplimented the medium to actived the signaling pathway after BMPR-IB gene silencing, the expression of receptors and Smads on repression or activation were investigated by real time RT-PCR. The results showed that the expression of BMPR-IA and BMPR-II was increased, while the expression of Smadl, Smad5 and Smad8 was decreased, and Smad7 was significanctly increased; the expression of BMPR-IA and BMPR-II was decreased by supplying BMP6 in the medium and the expression of Smadl, Smad5 and Smad8 were correspondingly increased while Smad7 was decreased; after the BMP signaling pathway was activated by BMP6, no significant changes were found in the expression of Smadl and Smad5 between the BMP6 groups and the blank, and Smad8 was decreased significantly, but no change was susgested, compared with interfered groups; furthermore, the expression of Smad7 were increased, regardless of supplying BMP6 or not in the medium.