YAP1在发情期不同繁殖力湖羊子宫内膜中的表达模式及功能分析

Expression Patterns and Functional Analysis of YAP1 in Endometrium of Hu Sheep with Different Fertility during Estrus

旨在分析Hippo信号通路的效应基因YAP1在不同繁殖力湖羊子宫内膜中的表达模式及功能。本研究根据系谱档案和BMPR-1B基因多态性分析,将9只体况相近且健康的经产母羊分为3组(HBB、LBB以及LB+,n=3)。提取不同繁殖力湖羊子宫内膜组织RNA,并反转录为cDNA,实时荧光定量PCR (RT-qPCR)分析Hippo信号通路中几个主效基因的表达模式。运用生物信息学方法分析YAP1在不同物种间的氨基酸同源性及其亲缘关系。体外分离湖羊子宫内膜基质细胞,利用RNA干扰和细胞转染技术体外干扰YAP1,采用实时荧光定量PCR (RT-qPCR)分析YAP1基因对子宫容受性相关基因的影响。利用流式细胞术分析干扰YAP1对细胞凋亡的影响,同时利用RT-qPCR和Western blot检测干扰YAP1后凋亡及线粒体相关基因的表达。结果显示,Hippo信号通路中YAP1、LATS1、MST1基因在不同繁殖力湖羊子宫内膜组织中的表达水平存在差异,其中YAP1基因在高繁殖力(HBB)湖羊子宫内膜中的表达量显著高于两组低繁殖力(LB+、LBB)湖羊;氨基酸同源性及系统进化树分析显示,绵羊YAP1蛋白与山羊和牛的同源性最高,亲缘关系最近;干扰YAP1基因后,子宫内膜基质细胞中容受性相关基因(FOXO1、PRL、VEGF、OPN、COX-2)的mRNA表达水平均发生了显著的变化。同时,子宫内膜基质细胞的凋亡率显著升高,凋亡相关基因Bax、BIM、p53、caspase3、caspase8、caspase9表达水平及Bax/Bcl-2的比值显著升高,Bcl-2表达水平显著下调。此外,线粒体功能相关基因(FIS1、DNM1L、MFN2、PPARGC1A、OPA1)mRNA表达水平也发生了显著变化。YAP1可能通过抑制细胞凋亡和稳定线粒体功能等途径建立良好的子宫内膜容受性。

The aim of this study was to analyze the expression patterns and function of the effector gene YAP1 in Hippo signaling pathway in endometrium of Hu sheep with different fertility. Based on the genealogical archives and polymorphism analysis of BMPR-1B, a total of 9 healthy pluriparous ewes were divided into 3 groups(HBB, LBB and LB+, n=3). RNA was extracted from endometrial tissues of Hu sheep with different fertility, and reversed transcript into cDNA. Real-time quantitative PCR(RT-qPCR) was used to analyze the expression of several major genes in the Hippo signaling pathway in endometrium of Hu sheep with different fertility. The amino acid homology and genetic relationship of YAP1 protein in different species were analyzed by bioinformatics methods. Hu sheep endometrial stromal cells(ESCs) were isolated in vitro, and the expression of YAP1 was downregulated by using RNAi and cell transfection techniques in vitro. The effect of YAP1 gene on the expression of uterine receptivity related genes was analyzed by RT-qPCR. Moreover, the effect of interfering YAP1 on cell apoptosis was analyzed by flow cytometry, and the expression of apoptosis and mitochondria related genes was analyzed by RT-qPCR and Western blot. The expression levels of YAP1, LATS1 and MST1 in Hippo signaling pathway were different in endometrium tissues of Hu sheep with different fertility, and the expression level of YAP1 gene in high fertility Hu sheep(HBB) endometrium was significantly higher than that in low fertility Hu sheep(LB+, LBB) endometrium. Amino acid homology and phylogenetic tree analysis showed that YAP1 protein of goat and cattle had the highest homology and the closest genetic relationship with sheep. The mRNA expression levels of receptivity related genes(FOXO1, PRL, VEGF, OPN, COX-2) in ESCs were significantly changed after interfering YAP1. Meanwhile, the apoptosis rate of ESCs was significantly increased, the expression levels of Bax, BIM, p53, caspase3, caspase8, caspase9, the ratio of Bax/Bcl-2 were upregulated, and expression levels of Bcl-2 was downregulated after YAP1 silencing. In addition, the mRNA expression levels of genes related to mitochondrial function(FIS1, DNM1L, MFN2, PPARGC1A, OPA1) were also significantly changed. YAP1 may establish good endometrial receptivity by inhibiting apoptosis and stabilizing mitochondrial function.