基于单细胞转录组测序的细粒棘球蚴感染小鼠肝T细胞异质性分析

MicroRNA differential expression profiles and their diagnostic value in the sheep infected with Echinococcus granulosus

目的 从单细胞水平探究细粒棘球蚴感染小鼠不同时期肝组织微环境细胞中T细胞亚型组成及其转录谱特征。方法 从课题组前期细粒棘球蚴感染后1个月(1只)、3个月(1只)和6个月(2只)的BALB/c小鼠和健康小鼠(1只,对照组)肝组织的单细胞转录组测序数据集(中国国家生物信息中心组学原始数据归档库:CRA008416)(https://ngdc.cncb.ac.cn/gsa)中提取测序数据进行质控。采用统一流形逼近与投影(UMAP)算法对单细胞群聚类进行可视化作图,采用共享最近邻相似度(SNN)聚类算法分析最优细胞分群。采用SingleR软件包基于immgen参考数据集对细胞亚群进行细胞类型注释。使用Seurat软件包的FindMarkers函数分析不同时期感染小鼠和对照组小鼠的调节性T细胞(Treg)和CD8+T细胞的差异表达基因(DEG)。利用基因本体论(GO)和京都基因与基因组百科全书(KEGG)分别对DEG进行功能富集分析和通路富集分析。结果 质控后获得37 760个细胞,人工优化后分为8种类型细胞。对T细胞重聚类后获得12个细胞群。注释后鉴定获得7种T细胞,包括CD4+初始T细胞、CD4+效应T细胞、Treg、CD8+初始T细胞、CD8+T细胞、增殖性T细胞、γδ T细胞。在细粒棘球蚴感染1个月后T细胞各亚群占比无明显变化,感染后3个月增殖性T细胞(11.91%,56/470)和Treg (13.40%,63/470)占比均高于对照组(3.51%, 38/1 082;4.34%, 47/1 082),感染后6个月CD8+T细胞占比(30.20%,1 145/3 791)高于对照组(15.43%, 167/1 082)。Treg在感染后3个月高表达肿瘤坏死因子-α诱导蛋白8(Tnfaip8)、Maf、伊卡洛斯家族锌指3 (Ikzf3)等维持Treg的基因;CD8+T细胞在感染后6个月高表达白细胞表面分化抗原40配体(Cd40lg)、类几丁质酶3 (Chil3)、分泌型磷蛋白1 (Spp1)等耗竭性基因。GO分析结果显示,感染后3个月Treg的DEG主要富集于转化生长因子β受体复合物组装、正调节T细胞活化、环磷酸腺苷介导的信号传导等通路;感染后6个月CD8+T细胞的DEG主要富集调节血管内皮生长因子受体、色氨酸分解代谢过程、细胞外基质细胞信号等通路。KEGG分析结果显示,感染后3个月Treg的DEG主要参与原发性免疫缺陷和Ras信号传导途径等通路;感染后6个月CD8+T细胞DEG主要参与脂肪酸代谢、谷胱甘肽代谢、叶酸代谢等通路。结论细粒棘球蚴感染后3个月、6个月小鼠的肝组织T细胞亚型存在差异,感染后3个月Treg占比增加,感染后6个月CD8+T细胞占比增加,Treg、CD8+T细胞的DEG及其主要富集的通路存在差异。

Objective To screen the miRNAs differential expression profiles in the serum from the sheep infected with Echinococcus granulosus and evaluate their potential values in the diagnosis of echinococcosis.Methods 10 sheep were divided into an infection group(5) and a control group(5). The E. granulosus infected-group was given 2 000 eggs per sheep by gavage, and the control group of sheep was given saline. Sixty days after infection, peripheral blood was collected from both groups, and total serum RNA was extracted. Small RNA high-throughput sequencing was used to identify and screen differentially expressed miRNAs. Five differentially expressed miRNAs were validated by real-time quantitative PCR(qRT-PCR). MedCale software was used to draw receiver operating characteristic(ROC) curves, calculate the area under the curve(AUC), and select miRNAs with potential diagnostic values(AUC ≥ 0.7). qRT-PCR was used to detect the relative transcription levels of miRNAs with potential diagnostic values in 15 serum samples of sheep infected with E. granulosus, and the AUC, sensitivity, and specificity were calculated.Target genes of the differentially expressed miRNAs were predicted using miRanda and RNA hybrid software, and gene ontology(GO) enrichment analysis and Kyoto encyclopedia of genes and genomes(KEGG) pathway enrichment analysis were conducted.Results Sequencing results identified a total of 26 differentially expressed miRNAs, including21 upregulated and 5 downregulated. The qRT-PCR results showed that the relative expression levels of five relatively abundant differentially expressed miRNAs in infection group, including oar-miR-191, oar-let-7a, oar-miR-150, oar-miR-26a, and oar-miR-21, were 2.22 ± 0.31, 2.12 ± 0.24, 2.42 ± 0.35, 2.09 ± 0.15, and 3.23 ± 0.83, respectively, which were higher than the control group(1.00 ± 0.11). There was a statistically significant difference in the relative expression levels of oar-miR-191, oar-let-7a, oar-miR-150, and oar-miR-26a compared to the control group(t = 3.960, 4.766,4.096, 9.126;all P < 0.05). ROC curve analysis revealed that the AUC of oar-let-7a, oar-miR-26a, and oar-miR-21 were all less than 0.7. the AUC of oar-miR-191 was 0.858, with a 95% confidence interval(95% CI) of 0.719-0.997(P <0.05), indicating its high diagnostic value, with a sensitivity of 71.43% and specificity of 85.71%. The AUC of oar-miR-150 was 0.738, with a 95% CI of 0.550-0.926(P < 0.05), indicating its diagnostic significance, with a sensitivity of53.33% and specificity of 86.67%. The results of the GO significance enrichment analysis showed that miRNA target genes with transcriptional levels increased by more than twice were mainly related to stress response, cell surface molecules, protein binding, and other functions. The KEGG pathway enrichment analysis results indicate that miRNA target genes with at least a two-fold increase in transcription levels are mainly enriched in key signalling pathways such as inflammation response, autophagy, and apoptosis.Conclusion The oar-miR-191 and oar-miR-150 screened in this study have good sensitivity and specificity in the diagnosis of E. granulosus infection, suggesting their potential as a biomarker for the diagnosis of echinococcosis.