产黑普氏菌刺激原代人口腔角质细胞的时间序列基因表达谱研究

The time series gene expression profiles of primary human oral keratinocytes stimulated by Prevotella melaninogenica

目的基于时间序列分析,研究产黑普氏菌(Prevotella melaninogenica,Pm)刺激原代人口腔角质形成细胞(primary human oral keratinocytes,pHOKs)后基因表达的时间趋势,以探究Pm与pHOKs相互作用的潜在机制。方法利用生物信息学方法分析pHOKs分别与Pm共培养4、24 h后的高通量测序结果,使用Mfuzz聚类算法将具有相似时间表达模式的基因划分聚类,并进行GO、KEGG以及STRING网络分析,进一步利用Cytoscape软件取交集得到枢纽基因(Hub基因)。采用实时荧光定量PCR检测Hub基因的表达。结果Pm刺激pHOKs 4、24 h分别有1456个和1386个差异表达基因。通过Mfuzz将其划分为3个聚类,其中簇1基因随时间延长其表达呈现下降的趋势,主要参与上皮细胞分化和上皮细胞向间充质细胞转化等生物学过程;簇2基因随时间延长其表达呈现上升的趋势,主要参与细菌感染、视黄醇代谢等过程;簇3基因表达在刺激前期上调、后期下调,主要参与细胞因子相关信号通路等过程。在PPI网络中筛选出簇1 Hub基因为VEGFA、GDNF;簇2中的Hub基因为PTGS2、ICAM1等;簇3中的Hub基因为IL-6、CCL2等。RT-qPCR检测结果显示,VEGFA、PTGS2、IFIT1、IRF1与测序数据中随着时间延长基因的表达趋势一致。结论本研究对Pm刺激pHOKs过程中相关生物学分子的动态模式进行了深度挖掘,发现与视黄醇代谢相关的基因以及负调控EMT的基因在OLP中异常表达,Pm入侵上皮细胞后可能通过一些适应性机制逃避免疫监视,该发现有助于进一步探究Pm在口腔扁平苔藓中的致病机理。

Objective To investigate the time trend of gene expression of primary human oral keratinocytes(pHOKs)stimulated with Prevotella melaninogenica(Pm),and to explore the potential mechanism of interaction between Pm and pHOKs.Methods The high-throughput sequencing results of pHOKs co-cultured with Pm after 4 h and 24 h were analyzed using bioinformatics methods.Mfuzz clustering algorithm was used to cluster genes with similar temporal expression patterns,and GO,KEGG and STRING network analysis were utilized.Hub genes were obtained using Cytoscape.RT-qPCR was used to detect the expression of Hub genes.Results A total of 1456 and 1386 differentially expressed genes(DEGs)in pHOKs were screened out after stimulated by Pm for 4 h and 24 h,respectively.These genes were divided into three clusters based on Mfuzz.The expression of cluster 1 genes showed a downward trend with time,which were mainly involved in biological processes such as epithelial cell differentiation and epithelial-to-mesenchymal transition.Cluster 2 genes showed an upward trend with time,which were mainly involved in bacterial infection and retinol metabolism.Cluster 3 genes were up-regulated in the early stage of stimulation and down-regulated in the late stage,which were mainly involved in cytokine-related signaling pathways.VEGFA and GDNF were selected as Hub genes in cluster 1 within the PPI network.Hub genes in cluster 2 were PTGS2,ICAM1,etc.Hub genes in cluster 3 were IL-6,CCL2,etc.The results of RT-qPCR showed that VEGFA,PTGS2,IFIT1,and IRF1 were consistent with the expression trend of the genes in the sequencing data over time.Conclusion The study has revealed the dynamic patterns of pHOKs stimulated by Pm,and suggests that genes related to retinol metabolism and genes negatively regulating EMT are abnormally expressed in oral lichen planus(OLP)and Pm may evade immune surveillance through some adaptive mechanisms after invading epithelial cells.It is helpful to further explore the pathogenic mechanism of Pm in OLP.