牙龈卟啉单胞菌影响牙龈上皮细胞miR-155-5p通过JAK/STAR信号通路作用牙周炎的机制  

Mechanism of Porphyromonas gingivalis affecting periodontitis induced by JAK/STAR signaling pathway in gingival epithelial cells miR-155-5p

作　　者：李莹[1] 张波[1] 赵紫婷[1] LI Ying;ZHANG Bo;ZHAO Zi-ting(Department of Stomatology,Lishui Central Hospital,Lishui,Zhejiang 323000,China;不详)

机构地区：[1]丽水市中心医院口腔科,浙江丽水323000

出　　处：《中国微生态学杂志》2021年第1期51-57,共7页Chinese Journal of Microecology

摘　　要：目的通过生物信息学对GEO数据库进行分析筛选miRNA后运用分子生物学手段验证并对机制进行深入探讨,为未来牙周炎治疗的生物标志物筛选及靶向治疗提供理论依据。方法通过生物信息学分析GEO数据库发现牙周炎患者中差异表达的miRNA。在DIANA生信预测网站中发现了与JAK/STAT信号传导方式有关的miRNA。随后,TargetScan被用于预测miRNA的靶mRNA,该mRNA不仅在牙周炎中差异表达,而且与JAK/STAT信号传导有关。利用基因集富集分析(GSEA)寻找与JAK/STAT信号转导途径紧密相关的基因集。通过实时定量PCR(qRT-PCR)和免疫印迹法(Western blot)检测在牙周炎中差异表达并与JAK/STAT信号有关的miRNA和mRNA的表达。通过免疫组织化学(IHC)观察组织中IL6ST的表达。通过双重荧光素酶测定法证实了miRNA和mRNA之间的关系。此外,采用细胞内氧化活性氧红色荧光检测试剂盒检测活性氧(ROS)的变化。结果在牙周炎患者中,与正常组织比较,MiR-155-5p被下调,mRNA IL6ST被上调且差异均具有统计学意义(t=9.1887、2.8521,P=0.0006、0.0157)。与对照组比较,miR-155-5p在P.gingivalis处理组表达情况出现明显下降且IL6ST在处理后表达量出现明显升高,差异均有统计学意义(t=2.1253、1.8520,P=0.0135、0.0157)。miR-155-5p具有IL6ST 3′非翻译区的靶结合位点。通过过表达miR-155-5p能够明显抑制JAK/STAT信号通路p-STAT3、p-JAK2、IL6ST蛋白的表达(t=1.9248、2.5308、3.1075,P=0.0231、0.0116、0.0100)。感染牙龈卟啉单胞菌后,细胞中的ROS产生增加(t=3.0512、9.6327,均P<0.01)。结论牙龈卟啉单胞菌可抑制miR-155-5p表达,激活GECs中的JAK/STAR信号,促进牙周炎的发生和发展。Objective To analyze and screen miRNAs in GEO database by using bioinformatics,verify and explore the mechanism using molecular biology methods,and provide theoretical basis for the screening of biomarkers and targeted therapy for periodontitis.Methods The differentially expressed miRNAs in patients with periodontitis were found through bioinformatics analysis of the GEO database.The miRNAs associated with JAK/STAT signaling were found on the DIANA Bios Prediction website.Subsequently,TargetScan was used to predict the target mRNA of the miRNA which not only differentially expressed in periodontitis,but also was related to JAK/STAT signaling.Gene set enrichment analysis(GSEA)was used to find a set of genes closely related to the JAK/STAT signal pathway.The expression of miRNAs and mRNAs differentially expressed in periodontitis and associated with JAK/STAT signaling were detected with real-time quantitative PCR(qRT-PCR)and Western blotting(Western blot).The expression of IL6 ST in tissues was observed using immunohistochemistry(IHC).The relationship between miRNA and mRNA was confirmed with dual luciferase assay.The changes in reactive oxygen species(ROS)were detected using an intracellular oxidative ROS red fluorescence detection kit.Results In patients with periodontitis,compared with normal tissues,miR-155-5 p was down-regulated,while mRNA IL6 ST was up-regulated;the difference was statistically significant(t=9.1887,2.8521;P=0.0006,0.0157).Compared with the control group,the expression of miR-155-5 p in P.gingivalis treatment group was significantly decreased and the expression of IL6 ST was significantly increased after treatment with statistically significant differences(t=2.1253,1.8520;P=0.0135,0.0157).miR-155-5 p had a target binding site for the 3′untranslated region of IL6 ST.Overexpression of miR-155-5 p can significantly inhibit the expression of p-STAT3,p-JAK2,and IL6 ST proteins in the JAK/STAT signaling pathway(t=1.9248,2.5308,3.1075;P=0.0231,0.0116,0.0100).After infection with P.gingivalis,the pr

关 键 词：牙周炎 牙龈卟啉单胞菌 微小RNA JAK/STAT信号通路 

分 类 号：R781.42[医药卫生—口腔医学]