差异表达microRNA参与刀豆蛋白A诱导的自身免疫性肝炎小鼠模型肝损伤的功能分析

郝健亨; 李振城; 刘莹; 侯艺文; 高艳; 苗宇船; 刘杨; 郝慧琴

doi:10.3969/j.issn.1001-5256.2021.06.028

差异表达microRNA参与刀豆蛋白A诱导的自身免疫性肝炎小鼠模型肝损伤的功能分析

DOI: 10.3969/j.issn.1001-5256.2021.06.028

山西中医药大学基础医学院，山西晋中 030619

基金项目:

山西省教育厅科技创新项目 (2020L0423);

山西中医药大学科技创新能力培育计划项目 (2020PY-JC-07);

山西省应用基础研究计划面上项目 (201901D111333);

基于炎症反应的重大疾病创新药物山西省重点实验室开放课题基金资助项目 (SXIDL-2018-07);

山西省卫生健康委科研课题计划 (2018002)

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：郝健亨负责数据分析，撰写论文；李振城、刘莹、侯艺文、高艳、苗宇船参与收集数据；郝慧琴、刘杨负责拟定写作思路，指导撰写文章并最后定稿。

附录1~4见二维码

详细信息

作者简介:
郝健亨(1993—)，男，主要从事自身免疫性肝炎的中西医结合治疗研究

通信作者:
刘杨，liuyang1980@sxtcm.edu.cn

郝慧琴，hhq@sxtcm.edu.cn

中图分类号: R575.1
计量
- 文章访问数: 696
- HTML全文浏览量: 120
- PDF下载量: 31
- 被引次数: 0
出版历程
- 收稿日期: 2020-11-27
- 录用日期: 2020-12-25
- 出版日期: 2021-06-20

A functional analysis of differentially expressed microRNAs involved in liver injury in mice with autoimmune hepatitis induced by concanavalin A

School of Basic Medical Sciences, Shanxi University of Chinese Medicine, Jinzhong, Shanxi 030619, China

摘要

摘要: 目的探讨了刀豆蛋白A(ConA)诱导的自身免疫性肝炎(AIH)小鼠模型肝损伤发生发展过程中差异表达microRNA(miRNA)的变化和潜在作用。方法健康雄性SPF级C57BL/6小鼠8只，随机分为模型组(n=4)和对照组(n=4)。模型组按15 mg/kg的剂量给予尾静脉注射ConA，对照组给予等剂量生理盐水，造模8 h后处死全部小鼠，提取肝组织总RNA，采用基因芯片筛选差异表达miRNA，并对表达上调及表达下调的miRNA进行靶基因预测和功能分析。两组间比较采用独立样本t检验鉴定差异表达的miRNA。结果主成分分析结果显示，基因芯片中所提取数据的组间差异满足进一步分析的条件。与对照组比较，模型组小鼠肝脏中共检测到31个上调和18个下调表达的miRNA，与959个(601个上调，358个下调)靶基因存在调控关系。GO分析显示，与对照组比较，模型组中表达上调miRNA的靶基因主要具备"DNA binding"(P值=1.47×10^-6)等分子功能，参与"transcription，DNA-templated"(P=2.36×10^-7) 等生物学过程，并主要富集在"neuronal cell body"(P=5.99×10^-6)等细胞组分。表达下调miRNA的靶基因主要具备"RNA polymerase Ⅱ proximal promoter sequence-specific DNA binding"(P=2.49×10^-6)等分子功能，参与"regulation of transcription, DNA-templated"(P=1.64×10^-11)等生物过程，并主要富集在"nucleoplasm"(P=4.30×10^-10)等细胞组分。KEGG通路分析结果表明上调靶点主要涉及"Endocytosis"(P=0.000 4)等，下调靶点主要涉及"Hippo信号通路"(P=0.004)。结论 AIH的发病中存在miRNA差异表达，这些miRNA差异表达谱可以为AIH的临床治疗提供新的靶点。
- 肝炎, 自身免疫性 /
- 微RNAs /
- 计算生物学
Abstract: Objective To investigate the changes and potential effects of differentially expressed microRNAs (miRNAs) in the development and progression of liver injury in a mouse model of autoimmune hepatitis (AIH) induced by concanavalin A (ConA). Methods Eight healthy male specific pathogen-free C57BL/6 mice were randomly divided into model group and control group, with four mice in each group. The mice in the model group were given tail vein injection of ConA 15 mg/kg, and those in the control group were given an equal volume of normal saline. All mice were sacrificed after 8 hours of modeling, Total RNA in liver tissue was extracted, gene microarray was used to screen out differentially expressed miRNAs, and target prediction and function analysis were performed for upregulated and downregulated miRNAs. The independent samples t-test was used for comparison of differentially expressed miRNAs between two groups. Results The principal component analysis showed that the inter-group difference of the data extracted by gene microarray met the conditions for further analysis. Compared with the control group, the model group had 31 upregulated miRNAs and 18 downregulated miRNAs in mouse liver, which had a regulatory relationship with 959 target genes (601 upregulated genes and 358 downregulated genes). GO analysis showed that in the model group, the target genes of the upregulated miRNAs mainly had the molecular functions such as "DNA binding" (P=1.47×10^-6), participated in the biological processes such as "transcription, DNA-templated" (P=2.36×10^-7), and were mainly enriched in the cellular components such as "neuronal cell body" (P=5.99×10^-6), while the target genes of the downregulated miRNAs had the molecular functions such as "RNA polymerase Ⅱ proximal promoter sequence-specific DNA binding" (P=2.49×10^-6), participated in the biological processes such as "regulation of transcription, DNA-templated" (P=1.64×10^-11), and were mainly enriched in the cellular components such as "nucleoplasm" (P=4.30×10^-10). KEGG pathway enrichment analysis showed that the target genes of the upregulated miRNAs were mainly enriched in "Endocytosis" (P=0.000 4), while the target genes of the downregulated miRNAs were mainly enriched in the "Hippo signaling pathway" (P=0.004), and the above functional analysis results were statistically significant (P < 0.05). Conclusion There are differentially expressed miRNAs in the pathogenesis of AIH, and these differentially expressed miRNAs can provide new targets for the clinical treatment of AIH.
- Hepatitis, Autoimmune /
- MicroRNAs /
- Computational Biology

HTML全文

图 1 PCA相关性分析结果

注：Model 1~4为模型组，sham 1~4为对照组。

下载: 全尺寸图片幻灯片

图 2 差异表达miRNA的火山分布图

注：红色和绿色虚线分别反映了上下表达的阈值(FC=2.0)，蓝色虚线表示P=0.05的界限。

下载: 全尺寸图片幻灯片

图 3 差异表达miRNA的聚类分析热图

注：Model 1~4为模型组；Control 1~4为对照组。

下载: 全尺寸图片幻灯片

图 4 差异表达miRNA靶基因GO富集分析结果

注：a，上调表达miRNA；b，下调表达miRNA。

下载: 全尺寸图片幻灯片

图 5 差异表达miRNA靶基因KEGG富集分析结果

注：a，上调表达miRNA；b，下调表达miRNA。

下载: 全尺寸图片幻灯片

表 1 PCR引物序列

miRNA	序列
mmu-miR- 21a-3p	R：5′-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGGACAGCCC-3′
	F：5′-ACACTCCAGCTGGGCAACAGCAGTCGAT-3′
mmu-miR- 1934-3p	R：5′-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGTCACCAGC-3′
	F：5′-ACACTCCAGCTGGGAGGATGACGGTGGGG-3′
mmu-miR- 188-5p	R：5′-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGCCCTCCA-3′
	F：5′-ACACTCCAGCTGGGCATCCCTTGCATGG-3′
mmu-miR- 22-5p	R：5′-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGTAAAGCTT-3′
	F：5′-ACACTCCAGCTGGGAGTTCTTCAGTGGC-3′
mmu-miR- 7055-3p	R：5′-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGCTGGTGGG-3′
	F：5′-ACACTCCAGCTGGGTTGCTACTTTGATAC-3′
mmu-miR- 126a-5p	R：5′-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGCGCGTAC-3′
	F：5′-ACACTCCAGCTGGGCATTATTACTTTTGG-3′
All	R：5′-TGGTGTCGTGGAGTCG-3′
U6	F：5′-CTCGCTTCGGCAGCACA-3′
	R：5′-AACGCTTCACGAATTTGCGT-3′

下载: 导出CSV

表 2 各组小鼠肝脏差异表达基因mRNA相对水平的比较

组别	mmu-miR-21a-3p	mmu-miR-1934-3p	mmu-miR-188-5p	mmu-miR-22-5p	mmu-miR-7055-3p	mmu-miR-126a-5p
对照组	1.03±0.31	1.03±0.45	1.18±0.29	-1.27±0.36	-1.2±0.21	-1.1±0.32
模型组	20.68±3.00	16.4±2.31	19.5±2.01	-3.48±0.80	-2.6±0.31	-4.3±1.08
t值	88.870	25.820	45.490	4.739	2.148	11.33
P值	＜0.05	＜0.05	＜0.05	＜0.05	＜0.05	＜0.05

下载: 导出CSV

参考文献(26)

[1]	KRAWITT EL. Autoimmune hepatitis[J]. N Engl J Med, 2006, 354(1): 54-66. DOI: 10.1056/NEJMra050408.
[2]	PAPE S, SCHRAMM C, GEVERS TJ. Clinical management of autoimmune hepatitis[J]. United European Gastroenterol J, 2019, 7(9): 1156-1163. DOI: 10.1177/2050640619872408.
[3]	FRANCQUE S, VONGHIA L, RAMON A, et al. Epidemiology and treatment of autoimmune hepatitis[J]. Hepat Med, 2012, 4: 1-10. DOI: 10.2147/HMER.S16321.
[4]	LOOSEN SH, SCHUELLER F, TRAUTWEIN C, et al. Role of circulating microRNAs in liver diseases[J]. World J Hepatol, 2017, 9(12): 586-594. DOI: 10.4254/wjh.v9.i12.586.
[5]	HAO JH, CHEN H, GAO Y, et al. Effects of Saikosaponin d on differentially expressed genes CTLA-4, IL-10 and IL-17 in mice with autoimmune hepatitis[J]. J Shanghai Jiaotong Univ (Med Sci), 2020, 40(3): 303-309. DOI: 10.3969/j.issn.1674-8115.2020.03.005. 郝健亨, 陈浩, 高艳, 等. 柴胡皂苷d对自身免疫性肝炎小鼠差异表达基因CTLA-4、IL-10和IL-17的影响[J]. 上海交通大学学报(医学版), 2020, 40(3): 303-309. DOI: 10.3969/j.issn.1674-8115.2020.03.005.
[6]	CHEN H, HAO JH, LI ZC, et al. Screening for differentially expressed genes in mice with autoimmune hepatitis and effect of saikosaponin-D on the expression of several differentially expressed genes[J]. J Clin Hepatol, 2020, 36(4): 840-846. DOI: 10.3969/j.issn.1001-5256.2020.04.026. 陈浩, 郝健亨, 李振城, 等. 自身免疫性肝炎小鼠模型差异表达基因的筛查及柴胡皂苷D对部分差异表达基因表达的影响[J]. 临床肝胆病杂志, 2020, 36(4): 840-846. DOI: 10.3969/j.issn.1001-5256.2020.04.026.
[7]	LIU Y, CHEN H, HAO JH, et al. Characterization and functional prediction of the microRNAs differentially expressed in a mouse model of concanavalin a-induced autoimmune hepatitis[J]. Int J Med Sci, 2020, 17(15): 2312-2327. DOI: 10.7150/ijms.47766.
[8]	SUCHER E, SUCHER R, GRADISTANAC T, et al. Autoimmune hepatitis-immunologically triggered liver pathogenesis-diagnostic and therapeutic strategies[J]. J Immunol Res, 2019, 2019: 9437043. DOI: 10.1155/2019/9437043.
[9]	THAN NN, JEFFERY HC, OO YH. Autoimmune hepatitis: Progress from global immunosuppression to personalised regulatory T cell therapy[J]. Can J Gastroenterol Hepatol, 2016, 2016: 7181685. Doi: 10.1155/2016/7181685.
[10]	HAYES CN, CHAYAMA K. MicroRNAs as biomarkers for liver disease and hepatocellular carcinoma[J]. Int J Mol Sci, 2016, 17(3): 280. DOI: 10.3390/ijms17030280.
[11]	LU FB, CHEN DZ, CHEN L, et al. Attenuation of experimental autoimmune hepatitis in mice with bone mesenchymal stem cell-derived exosomes carrying microRNA-223-3p[J]. Mol Cells, 2019, 42(12): 906-918. DOI: 10.14348/molcells.2019.2283.
[12]	BLAYA D, AGUILAR-BRAVO B, HAO F, et al. Expression of microRNA-155 in inflammatory cells modulates liver injury[J]. Hepatology, 2018, 68(2): 691-706. DOI: 10.1002/hep.29833.
[13]	SUI C, ZHANG L, HU Y. MicroRNA-let-7a inhibition inhibits LPS?induced inflammatory injury of chondrocytes by targeting IL6R[J]. Mol Med Rep, 20(3): 2633-2640. DOI: 10.3892/mmr.2019.10493.
[14]	RUSSELL JO, MONGA SP. Wnt/β-catenin signaling in liver development, homeostasis, and pathobiology[J]. Annu Rev Pathol, 2018, 13: 351-378. DOI: 10.1146/annurev-pathol-020117-044010.
[15]	ZHOU D, YU Y, AN Y, et al. EGCG mediated Wnt/β-catenin signaling pathway improves liver damage in mice with immune hepatitis[J]. Anatomy Res 2020, 42 (1): 38-43. DOI: GDJP.0.2020-01-007 周丹, 于洋, 安洋, 等. 表没食子儿茶素没食子酸酯通过Wnt/β-catenin信号通路改善免疫性肝炎小鼠肝损伤[J]. 解剖学研究, 2020, 42(1): 38-43. DOI: GDJP.0.2020-01-007.
[16]	ZHAO L, JIN Y, DONAHUE K, et al. Tissue repair in the mouse liver following acute carbon tetrachloride depends on injury-induced Wnt/β-catenin signaling[J]. Hepatology, 2019, 69(6): 2623-2635. DOI: 10.1002/hep.30563.
[17]	SONG H, DU C, WANG X, et al. MicroRNA-101 inhibits autophagy to alleviate liver ischemia/reperfusion injury via regulating the mTOR signaling pathway[J]. Int J Mol Med, 2019, 43(3): 1331-1342. DOI: 10.3892/ijmm.2019.4077.
[18]	LEI Y, WANG QL, SHEN L, et al. MicroRNA-101 suppresses liver fibrosis by downregulating PI3K/Akt/mTOR signaling pathway[J]. Clin Res Hepatol Gastroenterol, 2019, 43(5): 575-584. DOI: 10.1016/j.clinre.2019.02.003.
[19]	SZEKERCZÉS T, GÓGL A, ILLYÉS I, et al. Autophagy, mitophagy and microRNA expression in chronic hepatitis C and autoimmune hepatitis[J]. Pathol Oncol Res, 2020, 26(4): 2143-2151. DOI: 10.1007/s12253-020-00799-y.
[20]	SCHROEDER B, MCNIVEN MA. Importance of endocytic pathways in liver function and disease[J]. Compr Physiol, 2014, 4(4): 1403-1417. DOI: 10.1002/cphy.c140001.
[21]	WANG HX, LIU M, WENG SY, et al. Immune mechanisms of Concanavalin A model of autoimmune hepatitis[J]. World J Gastroenterol, 2012, 18(2): 119-125. DOI: 10.3748/wjg.v18.i2.119.
[22]	HONG L, CAI Y, JIANG M, et al. The Hippo signaling pathway in liver regeneration and tumorigenesis[J]. Acta Biochim Biophys Sin (Shanghai), 2015, 47(1): 46-52. DOI: 10.1093/abbs/gmu106.
[23]	MOHSENI R, KARIMI J, TAVILANI H, et al. Carvacrol ameliorates the progression of liver fibrosis through targeting of Hippo and TGF-β signaling pathways in carbon tetrachloride (CCl₄)-induced liver fibrosis in rats[J]. Immunopharmacol Immunotoxicol, 2019, 41(1): 163-171. DOI: 10.1080/08923973.2019.1566926.
[24]	SUI M, JIANG X, CHEN J, et al. Magnesium isoglycyrrhizinate ameliorates liver fibrosis and hepatic stellate cell activation by regulating ferroptosis signaling pathway[J]. Biomed Pharmacother, 2018, 106: 125-133. DOI: 10.1016/j.biopha.2018.06.060.
[25]	KONG Z, LIU R, CHENG Y. Artesunate alleviates liver fibrosis by regulating ferroptosis signaling pathway[J]. Biomed Pharmacother, 2019, 109: 2043-2053. DOI: 10.1016/j.biopha.2018.11.030.
[26]	LYBEROPOULOU A, CHACHAMI G, GATSELIS NK, et al. Low serum hepcidin in patients with autoimmune liver diseases[J]. PLoS One, 2015, 10(8): e0135486. DOI: 10.1371/journal.pone.0135486.