基于miRNA-mRNA调控网络筛选与验证参与Graves病B细胞增殖的关键基因  

Screening and validating potential key genes for B cell proliferation in Graves’ disease based on miRNA-mRNA regulatory network

作　　者：王玮 杨倩 王凯 钟惠敏 潘凡凡 查兵兵(指导) WANG Wei;YANG Qian;WANG Kai;ZHONG Huimin;PAN Fanfan;ZHA Bingbing(Department of Endocrinology,Shanghai Fifth People's Hospital,Fudan University,Shanghai 200240,China)

机构地区：[1]复旦大学附属上海市第五人民医院内分泌科,上海200240

出　　处：《中国免疫学杂志》2022年第22期2770-2776,共7页Chinese Journal of Immunology

基　　金：上海市自然科学基金项目(18ZR1429900,22ZR1448700);上海市医学重点专科建设计划项目(ZK2019B15)。

摘　　要：目的:利用生物信息学筛选出Graves病(GD)患者外周血B细胞中差异表达的miRNA,构建miRNA-mRNA调控网络,为GD治疗提供新靶点。方法:采用微阵列技术检测初发GD患者和健康受试者外周血CD19+B细胞中miRNA和mRNA表达谱。利用TargetScan和miRBase数据库对差异miRNA的靶基因进行预测,与差异mRNA取交集,筛选出目的靶基因。构建miRNA-mRNA差异表达网络,对其进行GO注释分析,筛选出与B细胞增殖相关的mRNA并采用荧光定量PCR(qRTPCR)以及流式细胞术(FACS)等手段进行验证。结果:GD患者外周血CD19+B细胞中有119个差异表达miRNA和572个差异表达mRNA,并筛选到159个潜在靶基因。通过构建miRNA-mRNA差异表达网络以及GO注释分析等手段,证实与B细胞增殖相关的CD79B在GD患者外周血CD19+B细胞中表达升高。qRT-PCR和FACS验证得出GD患者外周血CD19+B细胞中CD79B mRNA和蛋白水平均显著上调(P<0.01)。对调控CD79B的miRNA富集并进行qRF-PCR验证,结果显示GD患者外周血CD19+B细胞中hsa-miR-146b-5p表达显著下调(P<0.001)。利用TargetScan数据库预测发现CD79B mRNA的3’端非编码区存在潜在的hsa-miR-146b-5p结合位点。结论:GD患者外周血B细胞中hsa-miR-146b-5p的下调可能促进CD79B上调,引起B细胞活化与增殖。CD79B有望成为GD新的生物标志物和治疗的潜在靶点。Objective:To screen out the differentially expressed miRNAs in B cells in the peripheral blood of Graves’ disease(GD)patients by bioinformatics,and then to construct the miRNA-mRNA regulatory network,which will provide a new target for treating GD.Methods:Microarrays was used to analyze miRNA and mRNA expression profiles in CD19+B cells from the peripheral blood of healthy individuals and GD patients. Differentially expressed miRNA target genes by was predicted TargetScan and miRBase databases,which were intersected with differentially expressed mRNA,and the target genes were screened out. miRNA-mRNA regulatory network was constructed and analyzed by GO analysis,the mRNAs related to B cell proliferation were screened out and verified by real-time quantitative polymerase chain reaction(qRT-PCR)and flow cytometry(FACS).Results:There were 119 differentially expressed miRNA and 572 mRNA in CD19+B cells from the peripheral blood of GD patients,159 potential target genes were screened.Through the construction of miRNA-mRNA regulatory network and GO analysis,we confirmed that an elevated expression of CD79B associated with B cell proliferation in CD19+B cells from the peripheral blood of GD patients. qRT-PCR and FACS verification showed that CD79B mRNA and protein levels in CD19+B cells in peripheral blood of GD patients were significantly up-regulated(P<0.01).The expression of hsa-miR-146b-5p in CD19+B cells of peripheral blood of GD patients was significantly down-regulated(P<0.001).According to the prediction by TargetScan database,there was a putative hsa-miR-146b-5p binding site in the CD79B mRNA 3’UTR.Conclusion:The down-regulation of hsa-miR-146b-5p may promote the up-regulation of CD79B and thus lead to the activation and proliferation of B cells. CD79B is expected to be a new biomarker and a potential therapeutic target for GD.

关 键 词：GRAVES病 B细胞 MIRNA CD79B 微阵列 

分 类 号：R581.1[医药卫生—内分泌]