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慢性乙型肝炎与HBV相关慢加急性肝衰竭患者血浆外泌体差异miRNA的生物信息学分析
DOI: 10.3969/j.issn.1001-5256.2023.08.013
伦理学声明：本研究方案于2021年9月9日经由青岛市市立医院医学伦理委员会审批，批号：2021临审字Y第017号。所有研究对象已签署知情同意书。
利益冲突声明：本文不存在任何利益冲突。
作者贡献声明：张舒阳负责查阅、归纳文献，资料分析，撰写论文；毕研贞、刘守胜负责修改论文；柳盛负责参与血液标本收集；辛永宁负责拟定写作思路，指导撰写文章并最后定稿。
Differentially expressed microRNAs in plasma exosomes from patients with chronic hepatitis B or hepatitis B virus-related acute-on-chronic liver failure: A bioinformatics analysis
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摘要:
目的 探讨慢性乙型肝炎(CHB)患者与HBV相关慢加急性肝衰竭(HBV-ACLF)患者血浆外泌体microRNA(miRNA)表达谱差异，分析其功能及生物学过程，以期获得可用于HBV-ACLF临床诊断的参考依据。 方法 选取2021年10月—2022年6月青岛市市立医院感染科住院的6例CHB患者及青岛市第六人民医院血液净化中心接受治疗的6例HBV-ACLF患者。运用Illumina高通量测序技术对这些患者的血浆外泌体miRNA进行检测，筛选差异miRNA并进行功能富集分析，分析其参与的生物学过程。检测得到的外泌体差异miRNA以倍数上调＞2倍或下调＞2倍且P＜0.05为标准筛选。计量资料两组间比较采用Mann-Whitney U秩和检验。计数资料两组间比较采用χ2检验。 结果 筛选差异miRNA共249种，与CHB组相比，HBV-ACLF组上调miRNA 126种，下调miRNA 123种。生物信息学分析结果显示，这些差异表达miRNA主要参与了性腺发育、调控蛋白质稳定性、细胞对外界刺激反应等生物学过程，并与乙型肝炎、蛋白多糖在癌症中的作用、调节干细胞多能性、MAPK、Hippo、TNF、脂质代谢等信号通路密切相关。 结论 通过Illumina高通量测序技术筛选出的差异miRNA可能作为HBV-ACLF早期诊断及预后判断的生物标志物。 Abstract:Objective To investigate the differences in plasma exosomal microRNA (miRNA) expression profile between patients with chronic hepatitis B (CHB) and those with hepatitis B virus-related acute-on-chronic liver failure (HBV-ACLF), as well as their functions and biological processes, and to provide a reference for the clinical diagnosis of HBV-ACLF. Methods Six patients with CHB who were hospitalized in Department of Infectious Diseases, Qingdao Municipal Hospital, and six patients with HBV-ACLF who were treated in Blood Purification Center of Qingdao Sixth People's Hospital from October 2021 to June 2022 were enrolled. Illumina high-throughput sequencing was used to analyze the plasma exosomal miRNAs of these patients to obtain the differentially expressed miRNAs between the two groups. The differentially expressed miRNAs were screened, and a functional enrichment analysis was performed to identify the biological processes involving such miRNAs. The Mann-Whitney U rank sum test was used for comparison of continuous data between two groups, and the chi-square test was used for comparison of categorical data between groups. Results A total of 249 differentially expressed exosomal miRNAs were obtained according to the criteria of upregulation >2-fold or downregulation >2-fold, with P < 0.05, and compared with the CHB group, there were 126 upregulated miRNAs and 123 downregulated miRNAs. The bioinformatics analysis showed that these differentially expressed miRNAs were mainly involved in the biological processes such as gonadal development, regulation of protein stability, and cellular response to external stimuli, and they were closely associated with the signaling pathways such as hepatitis B, the role of proteoglycans in cancer, regulation of stem cell pluripotency, MAPK, Hippo, TNF, and lipid metabolism. Conclusion The differentially expressed miRNAs identified by Illumina high-throughput sequencing may be used as biomarkers for the early diagnosis and prognostic evaluation of HBV-ACLF. -
Key words:
- Hepatitis B, Chronic /
- Acute-On-Chronic Liver Failure /
- Exosomes /
- MicroRNAs
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图 1 CHB与HBV-ACLF血浆外泌体电镜下形态
注：a, CHB血浆外泌体电镜下形态；b, HBV-ACLF血浆外泌体电镜下形态。比例尺100 nm。
Figure 1. Electron microscopic morphology of plasma exosomes from CHB and HBV-ACLF
图 2 蛋白质免疫印迹法鉴定及纳米颗粒示踪分析结果
注：a, 外泌体特异性标志蛋白CD63、CD81、TSG101及内质网蛋白Calnexin的表达结果，1与2分别代表HBV-ACLF与CHB两组样本；b, 利用NTA分析CHB样本外泌体粒径的大小；c, 利用NTA分析HBV-ACLF样本外泌体粒径的大小。
Figure 2. Results of western blot and nanoparticle tracking analysis
图 3 差异miRNA的差异倍数和P值分布火山图
注：红色表示上调；绿色表示下调。
Figure 3. Volcano plot of FoldChange and P-value distribution of differentially expressed miRNA
图 4 样本间差异表达miRNA聚类分析热图(CHB vs HBV-ACLF)
Figure 4. The differentially expressed miRNA cluster analysis heat map(CHB vs HBV-ACLF)
表 1 两组患者一般资料比较
Table 1. Comparison of baseline clinical characteristics between CHB group and HBV-ACLF group
项目 CHB组(n=6) HBV-ACLF组(n=6) 统计值 P值 男性[例(%)] 4(66.67) 4(66.67) ＞0.05 年龄(岁) 56.00(48.75~65.25) 55.50(50.50~66.00) Z=-0.241 0.810 ALT(U/L) 23.94(15.47~36.38) 487.00(197.25~866.25) Z=-2.882 0.004 AST(U/L) 23.09(19.63~32.06) 249.00(194.75~692.00) Z=-2.882 0.004 TBil(μmol/L) 13.05(10.28~18.63) 284.90(268.08~355.93) Z=-2.882 0.004 DBil(μmol/L) 2.92(2.53~3.64) 209.20(184.03~226.58) Z=-2.882 0.004 WBC(×109/L) 4.92(4.09~5.62) 5.46(4.35~8.08) Z=-0.801 0.423 RBC(×1012/L) 4.56(4.30~4.73) 4.17(3.72~4.85) Z=-0.642 0.521 PLT(×109/L) 164.00(144.75~202.50) 86.00(50.25~148.75) Z=-2.082 0.037 PT(s) 12.50(11.53~13.55) 23.25(17.90~38.30) Z=-2.882 0.004 APTT(s) 31.15(28.63~39.25) 42.35(35.03~53.08) Z=-2.082 0.037 注：APTT，活化部分凝血活酶时间。 
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表 2 HBV-ACLF患者差异表达的miRNA
Table 2. Differentially expressed miRNAs in HBV-ACLF patients
上调miRNA 差异倍数 P值 下调miRNA 差异倍数 P值 hsa-miR-6886-3p 43.586 0.000 00 hsa-miR-3689b-5p 0.119 0.010 19 hsa-miR-6880-3p 42.256 0.000 00 hsa-miR-1298-5p 0.115 0.000 01 hsa-miR-10398-5p 41.267 0.000 00 hsa-miR-4754 0.115 0.000 01 hsa-miR-122b-3p 19.775 0.000 00 hsa-miR-183-3p 0.112 0.003 83 hsa-miR-411-3p 12.467 0.000 02 hsa-miR-449b-5p 0.106 0.000 38 hsa-miR-4327 11.427 0.000 15 hsa-miR-4317 0.106 0.015 70 hsa-miR-7111-3p 10.981 0.000 04 hsa-miR-5689 0.103 0.000 00 hsa-let-7b-3p 10.785 0.000 01 hsa-miR-520f-5p 0.098 0.010 06 hsa-miR-335-3p 10.718 0.000 07 hsa-miR-4291 0.089 0.001 71 hsa-miR-4646-5p 10.647 0.000 01 hsa-miR-96-3p 0.085 0.001 01 hsa-miR-6887-5p 10.511 0.000 05 hsa-miR-631 0.081 0.000 06 hsa-miR-1281 10.327 0.000 04 hsa-miR-3678-3p 0.066 0.000 00 hsa-miR-4510 9.819 0.000 00 hsa-miR-6755-5p 0.066 0.001 92 hsa-miR-2114-3p 9.280 0.000 14 hsa-miR-6841-5p 0.050 0.000 00 hsa-miR-6760-5p 8.961 0.000 02 hsa-miR-4795-5p 0.049 0.000 34
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慢性乙型肝炎与HBV相关慢加急性肝衰竭患者血浆外泌体差异miRNA的生物信息学分析.pdf
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