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肝泡型包虫病特异microRNA的筛选及初步研究
DOI: 10.3969/j.issn.1001-5256.2021.01.027
利益冲突声明：本研究不存在研究者、伦理委员会成员、受试者监护人以及与公开研究成果有关的利益冲突。
作者贡献声明：任宾、任利负责确诊病例的收集，RNA的提取和文章的撰写; 樊海宁、王海久负责统计学分析和文章的修改。
Screening and a preliminary study of specific microRNA for hepatic alveolar echinococcosis
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摘要:
目的 肝泡型包虫病由多房棘球蚴感染造成，通过对肝泡型包虫病患者组织和血浆中差异表达的miRNA进行筛查，寻找肝泡型包虫病新的生物标志物。 方法 纳入2016年6月—2018年5月在青海大学附属医院确诊的肝泡型包虫病患者，选取2例肝泡型包虫病患者的病灶边缘组织和3例临近病灶边缘的正常组织，以及3例肝泡型包虫病患者和3例健康对照者的血浆样本，使用Agilent Human miRNA芯片检测组织和血浆的miRNA表达谱，根据差异倍数(FC＞1.2)和P值(P＜0.05)筛选差异表达的miRNA，根据差异miRNA的靶基因预测结果，结合文献报道，选择与肝脏疾病相关的血浆miRNA和组织miRNA进行实时荧光定量PCR(qRT-PCR)验证。计量资料2组间比较采用t检验，相关性分析采用Spearman相关分析。 结果 肝泡型包虫病患者中的microRNA表达谱与健康人相比有显著不同，qRT-PCR验证发现6个microRNA中有3个miRNA(hsa-miR-4644，hsa-miR-136-5p，hsa-miR-483-3p)在肝泡型包虫病患者中显著差异表达(P＜0.05)。其中hsa-miR-4644和hsa-miR-483-3p在肝泡型包虫病患者中显著上调表达(P值均＜0.05)，hsa-miR-136-5p显著下调表达(P＜0.05)。通过TargetScan，PITA，microRNAorg数据库对差异miRNA进行靶基因预测，对三个数据库预测到的靶基因取交集，共有137个基因和miRNA之间有靶向关系。差异的hsa-miR-483-3p靶向调控参与人体免疫反应及与肝脏疾病有关的基因(IL-17A，IL-5，CD40LG，TAP2，TNF)。通过GO与KEGG分析发现，hsa-miR-483-3p的靶基因在免疫缺陷(Primary immunodeficiency)信号通路，IL-17信号通路，TNF信号通路中起重要作用。 结论 肝泡型包虫病有独特的microRNA表达谱，其中hsa-miR-483-3p可作为肝泡型包虫病的一种新的生物学标志物，其调控的靶基因主要参与Primary immunodeficiency信号通路，IL-17信号通路，TNF信号通路。但这些miRNA与肝泡型包虫病之间的调控关系有待进一步验证。 Abstract:Objective To investigate new biomarkers for hepatic alveolar echinococcosis by screening out differentially expressed microRNAs (miRNAs) in the tissues and plasma of patients with hepatic alveolar echinococcosis, since hepatic alveolar echinococcosis is caused by the infection of multilocular hydatid cyst. Methods Patients with hepatic alveolar echinococcosis diagnosed in Qinghai University Affilrated Hospital from June 2016 to May 2018 were in cluded. Two marginal tissue samples and three adjacent normal tissue samples were collected from patients with hepatic alveolar echinococcosis, and plasma samples were collected from three patients with hepatic alveolar echinococcosis and three healthy controls. Agilent Human miRNA microarray was used to obtain the miRNA expression profile in tissue and plasma, and differentially expressed miRNAs were screened out based on fold change (FC > 1.2) and P value (P < 0.05). Plasma miRNAs and tissue miRNAs associated with liver diseases were selected based on target gene prediction of differentially expressed miRNAs and literature reports, and quantitative real-time PCR (qRT-PCR) was used for validation. The t-test was used for comparison of continuous data between two groups. A spearman analysis was used to investigate correlcction. Results There was a significant difference in microRNA expression profile between the patients with hepatic alveolar echinococcosis and the health individuals, and qRT-PCR found that three miRNAs (hsa-miR-4644, hsa-miR-136-5p, hsa-miR-483-3p) were significantly differentially expressed in patients with hepatic alveolar echinococcosis (P < 0.05), among which hsa-miR-4644 and hsa-miR-483-3p were significantly upregulated (P < 0.05) and hsa-miR- 136-5p was significantly downregulated (P < 0.05) in patients with hepatic alveolar echinococcosis. Target gene prediction was performed for miRNAs based on TargetScan, PITA, and microRNAorg databases, and the intersection of the target genes predicted by these three databases showed that 137 genes were targeted with miRNAs. The differentially expressed miRNA hsa-miR-483-3p was involved in the target regulation of the genes (IL17A, IL5, CD40LG, TAP2, and TNF) associated with immune response and liver diseases. Gene ontology and Kyoto Encyclopedia of Genes and Genome analyses showed that the target genes of hsa-miR-483-3p played an important role in the primary immunodeficiency signaling pathway, the IL-17 signaling pathway, and the TNF signaling pathway. Conclusion Hepatic alveolar echinococcosis has a unique microRNA expression profile, among which hsa-miR-483-3p can be used as a new biomarker for hepatic alveolar echinococcosis, and the target genes regulated by this miRNA are mainly involved in the primary immunodeficiency signaling pathway, the IL-17 signaling pathway, and the TNF signaling pathway. However, further studies are needed to verify the regulatory relationship between these miRNAs and hepatic alveolar echinococcosis. -
Key words:
- Echinococcosis, Hepatic /
- MicroRNAs /
- Gene Expression Profiling
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图 1 miRNA表达谱中差异miRNA的火山图
注：a, 血浆样本; b, 组织样本。灰色点，P＞0.05的miRNA; 绿色点，log2(差异倍数)绝对值＜1.2但P＜0.05的miRNA; 红色点，log2(差异倍数)绝对值＞1.2且P＜0.05显著性上调的差异miRNA; 蓝色点，log2(差异倍数)绝对值＞1.2且P＜0.05显著性下调的差异miRNA。
表 1 AE患者miRNA表达谱中的差异miRNA
样本类型 基因名称 P值 差异倍数 趋势 mirbase编号 血浆 hsa-miRNA-136-5p ＜0.001 16.725 下调 MIMAT0000448 hsa-miRNA-4698 ＜0.001 22.626 下调 MIMAT0019793 hsa-miRNA-4644 0.044 10.077 上调 MIMAT0019704 hsa-miRNA-4306 0.026 2.150 下调 MIMAT0016858 hsa-miRNA-483-3p 0.032 3.877 上调 MIMAT0002173 组织 hsa-miRNA-1237-3p 0.016 10.928 上调 MIMAT0005592 hsa-miRNA-4306 0.015 41.615 下调 MIMAT0016858 hsa-miRNA-483-3p 0.045 8.868 上调 MIMAT0002173 
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表 2 血浆中差异miRNA水平影响分析
组别 例数 miRNA-136-5p miRNA-4698 miRNA-4644 miRNA-4306 miRNA-483-3p 健康组 15 1.017±0.189 1.137±0.640 1.030±0.271 1.048±0.317 1.012±0.165 AE组 15 0.485±0.171 0.748±0.372 1.642±0.718 0.840±0.285 2.213±1.294 t值 8.084 2.033 -3.091 1.891 -3.563 P值 ＜0.001 0.052 0.006 0.069 0.003
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表 3 AE组患者组织中差异miRNA水平影响分析
组别 例数 miRNA-1237-3p miRNA-4306 miRNA-483-3p 正常组织 15 1.022±0.226 1.022±0.214 1.007±0.123 病灶边缘组织 15 1.165±0.249 0.919±0.185 1.941±0.907 t值 -1.643 1.418 -3.952 P值 0.112 0.167 0.001
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