牛黄体细胞与卵泡颗粒细胞差异基因的筛选

Screening of differential genes between bovine corpus luteum cells and follicular granulosa cells

旨在探究牛卵巢大黄体细胞(LLCs)和颗粒细胞(GCs)之间的差异表达基因,筛选颗粒细胞黄体化密切相关的基因。在GEO数据库获得GSE83524芯片数据,通过R软件limma包中的DESeq2来筛选牛优势卵泡GCs和卵巢黄体LLCs中的差异表达基因;通过DAVID软件对获得的差异表达基因进行GO分析和KEGG信号通路分析;使用String软件构建差异表达基因之间的蛋白互作网络(PPI);将互作数据导入Cytoscape软件对差异表达基因进行可视化分析,同时利用软件自带Cyto-Hubba插件的MCC算法筛选连通度最大的前10位的关键基因;最后通过实时荧光定量PCR对筛选出的与GCs增殖及其黄体化相关基因进行验证。通过R软件limma包中的DESeq2分析后,在2种细胞中共获得242个差异表达基因,其中上调表达基因156个,下调表达基因86个;GO功能富集分析显示,生物学过程占46.97%,细胞组分占31.82%,分子功能占21.21%;KEGG共发现16条通路,其中FOXO信号通路与GCs的增殖、卵泡的发育、闭锁和黄体化密切相关。PPI网络互作分析,筛选出了10个连通度最大的关键基因;实时荧光定量PCR结果表明,PRLR、SPARCL1、INHA、GREB1、CYP19A1在LLCs和GCs中的表达趋势与GEO芯片数据结果一致,且PRLR在LLCs中的表达量极显著地高于GCs(P<0.01),SPARCL1在LLCs中的表达量显著高于GCs(P<0.05);GREB1、CYP19A1在GCs中的表达量极显著地高于LLCs(P<0.01),INHA在GCs中的表达量显著高于LLCs(P<0.05)。本研究推测这些基因在牛卵巢中对GCs黄体化起重要作用,为深入研究细胞特异性及GCs细胞向LLCs的分化机制奠定基础。

This study focuses on exploring the differentially expressed genes between Large luteum cells(LLCs)and granulosa cells(GCs)and screening the genes closely related to the granulosa cells luteinization of bovine ovaries.GSE83524chip data was obtained from GEO database.DESeq2 in R limma package was used to screen differentially expressed genes from dominant follicular GCs and LLCs of the corpus luteum in bovine.DAVID software was used to analyze the GO and KEGG signal pathways of the differentially expressed genes.String software was used to construct the protein interaction network(PPI)between differentially expressed genes.The interaction data were imported into Cytoscape software for visual analysis of differentially expressed genes,and the MCC algorithm with cyto-Hubba plug-in was used to screen the top 10hub genes with the highest connectivity.Finally,the selected genes related to the proliferation and luteinization of granulocytes were verified by real-time fluorescence quantitative PCR.After DESeq2analysis in R limma package,242differentially expressed genes were obtained from the two types of cells,among which 156genes were up-regulated and 86genes were down-regulated.GO analysis showed that biological processes accounted for 46.97%;cell component accounted for 31.82%;molecular function accounted for 21.21%.KEGG found 16pathways,among which FOXO signaling pathway was closely related to granulosa cell proliferation,follicular development,atresia,and luteinization.Ten hub genes with the highest connectivity were selected by PPI network interaction analysis.The expression trend of PRLR,SPARCL1,INHA,GREB1and CYP19A1in LLCs and GCs was consistent with the data of GEO chip,and the expression of PRLRin LLCs was extremely significantly higher than that of GCs(P<0.01),SPARCL1expression in LLCs was significantly higher than that in GCs(P<0.05);the expression of GREB1and CYP19A1in GCs was extremely significantly higher thanthat of LLCs(P<0.01),the expression of INHAin GCs was significantly higher than that of LLCs(P<0.05).In this study we speculate that these genes play an important role in the lutealization of granulosa cells in bovine ovaries,which lays a foundation for in-depth study of cell specificity and the differentiation mechanism of GCs into LLCs.