牛优势卵泡膜细胞黄体化相关基因的筛选

Screening of genes related to luteinization in dominant follicular theca cells of cattle

【目的】探究牛卵巢小黄体细胞(SLCs)和膜细胞(TCs)中的差异表达基因,进而筛选与TCs黄体化密切相关的基因,为深入研究SLCs、TCs特异性及TCs向SLCs分化的机制提供参考。【方法】利用R软件limma包中的DESeq2,筛选GEO芯片数据库GSE83524数据集TCs和SLCs中的差异表达基因,再用goseq软件对筛选出的差异表达基因进行GO功能富集分析,用kobas软件进行KEGG信号通路分析,使用String结合Cytoscape软件进行PPI网络互作分析。采集思南黄牛颗粒细胞(GCs)和TCs,以RPLP0作为内参基因,用荧光定量PCR法对筛选出的与TCs增殖及黄体化相关的基因进行验证。【结果】共筛选获得237个差异表达基因,其中表达上调的基因115个,表达下调的基因122个。GO功能分析结果显示,参与生物学过程的有147个基因,占差异表达基因总数的53.6%,分别富集于30个组;参与细胞组分的有125个基因,占差异表达基因总数的21.4%,分别富集于12个组;参与分子功能的有98个基因,占差异表达基因总数的25.0%,分别富集于14个组。KEGG分析共发现17条通路,其中可能与卵泡内膜细胞黄体化相关的通路为FSHR、PLA2G4A、CYP19A1、CYP17A1等4个基因参与的卵巢类固醇生成通路。经过PPI网络互作分析,筛选出了前10位的hub基因。荧光定量PCR分析结果显示,FSHR、PLA2G4A、CYP19A1、CYP17A1、BUB1B和KIF20A在SLCs和TCs中的转录水平与GEO芯片数据结果一致,表现为FSHR、CYP19A1和CYP17A1在TCs中的表达量极显著高于其在SLCs中的表达量(P<0.01),PLA2G4A、BUB1B和KIF20A在TCs中的表达量亦显著高于其在SLCs中的表达量(P<0.05)。【结论】差异表达基因FSHR、PLA2G4A、CYP19A1、CYP17A1、BUB1B和KIF20A在牛优势卵泡TCs增殖分化形成黄体的过程中发挥关键作用。

【Objective】This study explored differentially expressed genes in small luteum cells(SLCs)and theca cells(TCs)of cattle ovary and screened genes related to TCs lutealization to provide reference for further study of SLCs,TCS specificity and TCS differentiation to SLCs.【Method】DESeq2 of limma package in R was used to screen differentially expressed genes between TCs and SLCs in the GSE83524 of GEO database.GO function enrichment was analyzed using the goseq software and the kobas software was used to perform KEGG signal pathway analysis.Protein-protein interaction network(PPI)was constructed through String combined with the Cytoscape software,and the genes related to TCs proliferation and luteinization were verified by real-time PCR.【Result】A total of 237 differentially expressed genes were obtained,among which 115 were up-regulated and 122 were down-regulated.GO functional analysis showed that 147 genes were involved in biological processes,accounting for 53.6%of total differentially expressed genes,and enriched in 30 groups.There were 125 genes involved in cell components,accounting for 21.4%of total differentially expressed genes,enriched in 12 groups.There were 98 genes involved in molecular function,accounting for 25.0%of total differentially expressed genes,enriched in 14 groups.A total of 17 pathways were found through KEGG signaling pathway analysis,among which FSHR,PLA2G4A,CYP19A1 and CYP17A1 were involved in ovarian steroidogenesis.Top 10 hub genes were screened by PPI network interaction analysis.Real-time PCR results showed that transcription levels of FSHR,PLA2G4A,CYP19A1,CYP17A1,BUB1B and KIF20A in SLCs and TCs were consistent with the GEO data,the mRNA amounts of FSHR,CYP19A1 and CYP17A1 in TCs were extremely significantly greater than those of SLCs(P<0.01)and the mRNA amounts of PLA2G4A,BUB1B and KIF20A in TCs were significantly greater than those of SLCs(P<0.05).【Conclusion】Differentially expressed genes of FSHR,PLA2G4A,CYP19A1,CYP17A1,BUB1B and KIF20A played a key role in the proliferation and differentiation of bovine dominant follicular TCs to luteum formation.