基于RNA-Seq分析挖掘唾液腺多形性腺瘤核心基因及通路

Identification of core differentially expressed genes and pathways of salivary gland pleomorphic adenoma based on RNA-Seq

目的:筛选多形性腺瘤(pleomorphic adenoma,PA)与瘤旁唾液腺腺体间的差异表达基因,挖掘PA形成过程中的核心基因及通路。方法:采用RNA-Seq技术,检测5例PA患者的配对肿瘤与瘤旁唾液腺腺体,筛选2组间差异基因。运用蛋白互作数据库STRING分析预测差异基因所编码蛋白间的相互作用。筛选互作网络中的核心模块及核心基因,分析核心模块中激活的信号通路,预测上述模块与PLAG1可能的相互作用关系。RT-PCR验证核心基因在20例PA患者的配对肿瘤组织及瘤旁唾液腺中的表达。采用SPSS 20.0软件包对数据进行统计学分析。结果:共测得3810个差异基因,其中2021个下调,1789个上调。核心模块中存在PI3K-AKT信号通路、ER受体信号通路、Rap1信号通路、cGMP-PKG信号通路的激活。PLAG1可能通过PHLPP1、LRRK2、β-catenin蛋白与核心模块发生作用。RTPCR显示,核心基因在20例PA样本及其瘤旁唾液腺组织中的差异趋势与测序结果一致,且具有统计学意义(P<0.0001)。结论:核心基因ADCY3、 ADCY5、 ADORA1、 APLN、 APP、 C5、 CCL28、 DRD2、 FPR3、 GABBR1可能在PA发生、发展过程中起着重要作用,可为预防PA复发及预后标志物筛选提供思路。PLAG1可能通过PHLPP1、LRRK2、β-catenin间接激活PI3K-AKT信号通路、ER受体信号通路、Rap1信号通路、cGMP-PKG信号通路,从而诱发PA形成。

PURPOSE:The aim of this study was to identify the core differentially expressed genes(DEGs)signatures during pleomorphic adenoma(PA)and identified the key pathways and genes in PA.METHODS:The mRNA expression of 5 cases of PA and salivary gland was detected by RNA-Seq.Then the differentially expressed genes(DEGs)were screened.Protein-protein interaction(PPI)network of the DEGs was constructed and the core modules and hub genes were screened.The activated pathways associated with the core modules was analyzed,and the possible interaction between them and PLAG1 was predicted.Furthermore,the hub genes expression was validated by RT-PCR in 20 pairs of PA and normal salivary tissues.Statistical analysis was performed using SPSS 20.0 software package.RESULTS:In total,3810 DEGs were identified in PA,including 2021 down-regulated genes and 1789 up-regulated genes.PI3K-AKT signaling pathway,ER receptor signaling pathway,Rap1 signaling pathway and cGMP-PKG signaling pathway were activated in the core modules.PLAG1 may interact with the core modules through PHLPP1,LRRK2,and beta-catenin proteins.The expression of the hub genes in 20 pairs of PA and normal salivary tissues were validated by RT-PCR,the expression trend was in accordance with the mRNA sequencing(P<0.0001).CONCLUSIONS:Hub genes including ADCY3,ADCY5,ADORA1,APLN,APP,C5,CCL28,DRD2,FPR3,GABBR1 may play important roles in the oncogenesis and development of PA,which can provide ideas for preventing recurrence and screening prognostic markers of PA.PLAG1 may induce oncogenesis of PA by interacting with PHLPP1,LRRK2,and beta-catenin proteins,which indirectly activate the PI3K-AKT signal pathway,ER receptor signal pathway,Rap1 signal pathway,and cGMP-PKG signal pathway.