慢加急性肝衰竭不同预后患者血浆外泌体差异蛋白的生物信息学分析

焦彦; 张莹; 时红林; 卢旺; 陈德喜; 陈煜; 时红波

doi:10.3969/j.issn.1001-5256.2021.04.022

慢加急性肝衰竭不同预后患者血浆外泌体差异蛋白的生物信息学分析

DOI: 10.3969/j.issn.1001-5256.2021.04.022

焦彦^{1, 2},
张莹^{1, 2},
时红林^{1, 2, ,},
卢旺³,
陈德喜^{1, 2},
陈煜³,
时红波^{1, 2}

1.
首都医科大学附属北京佑安医院, 北京市肝病研究所, 北京 100069
2.
北京市肝炎与肝癌精准医疗及转化工程技术研究中心, 北京 100069
3.
首都医科大学附属北京佑安医院肝病中心四科, 肝衰竭与人工肝治疗研究北京市重点实验室, 北京 100069

基金项目:

北京市属医学科研院所公益发展改革试点项目 (京医研2019-6);

中国肝炎防治基金会王宝恩肝纤维化研究基金 (2020033);

中国肝炎预防控制基金会 (TQGB20190050);

国家自然科学基金 (81672026)

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: 焦彦负责实施研究过程和采集整理数据; 张莹、卢旺负责调研整理文献; 时红林负责技术或材料支持; 陈德喜负责修订论文; 陈煜负责患者入组及标本的收集整理; 时红波负责提出研究选题, 设计研究方案以及修订论文。

详细信息

作者简介:
焦彦(1993—), 女, 主要从事肝衰竭、肝炎方面的研究

通信作者:
时红波, shihongbo@ccmu.edu.cn

中图分类号: R575.3
计量
- 文章访问数: 708
- HTML全文浏览量: 211
- PDF下载量: 55
- 被引次数: 0
出版历程
- 收稿日期: 2020-10-13
- 录用日期: 2020-11-05
- 出版日期: 2021-04-20

A bioinformatics analysis of differentially expressed proteins in plasma exosome of acute-on-chronic liver failure patients with different prognoses

Yan JIAO^{1, 2},
Ying ZHANG^{1, 2},
Honglin SHI^{1, 2
, ,},
Wang LU³,
Dexi CHEN^{1, 2},
Yu CHEN³,
Hongbo SHI^{1, 2}

1.
Beijing Institute of Hepatology, Beijing YouAn Hospital, Capital Medical University, Beijing 100069, China
2.
Beijing Engineering Research Center for Precision Medicine and Transformation of Hepatitis and Liver Cancer, Beijing 100069, China
3.
Beijing Municipal Key Laboratory of Liver Failure and Artificial Liver Treatment Research, Fourth Department of Liver Disease, Beijing YouAn Hospital, Capital Medical University, Beijing 100069, China

摘要

摘要: 目的筛选不同预后慢加急性肝衰竭(ACLF)患者血浆外泌体中的差异蛋白, 分析其功能及生物学过程, 为患者临床诊断提供参考依据。方法前瞻性选取2019年7月—10月于首都医科大学附属北京佑安医院住院确诊的ACLF患者10例, 随访90 d, 患者死亡或肝移植归入肝移植/死亡组(n=5), 患者存活归入生存组(n=5), 两组一般资料指标比较采用Mann-Whitney U秩和检验。采用非标记(Label-free) 定量蛋白质组学技术对血浆外泌体蛋白进行鉴定和定量分析, 筛选差异蛋白并进行功能富集分析, 使用R-3.5.1软件对差异蛋白进行层次聚类分析, 分析其参与的生物学过程。结果外泌体蛋白质组学分析共鉴定出860种蛋白, 以倍数上调＞1.2倍或下调＞1.2倍且P＜0.05的标准筛选出差异表达蛋白116种, 与肝移植/死亡组相比, 生存组上调蛋白62种、下调蛋白54种。生物信息学分析结果显示, 这些蛋白主要参与了免疫反应、信号转导、囊泡介导的转运、细胞死亡和增殖等生物学过程, 并与炎症反应、糖类和氨基酸代谢、肝细胞损伤及再生等信号通路密切相关。结论非标记定量蛋白质组学技术筛选出的差异蛋白可能作为ACLF早期诊断及预后判断的血清学标志物。
- 慢加急性肝功能衰竭 /
- 外泌体 /
- 蛋白质组学 /
- 计算生物学
Abstract: Objective To investigate the differentially expressed proteins in the plasma exosome of acute-on-chronic liver failure (ACLF) patients with different prognoses, to analyze their functions and biological processes, and to provide a basis for clinical diagnosis. Methods A prospective study was performed for 10 ACLF patients who were hospitalized and diagnosed in Beijing YouAn Hospital, Capital Medical University, from July 2019 to October 2019, and the patients were followed up for 90 days. The patients who died or received liver transplantation were enrolled as liver transplantation/death group (5 patients), and the patients who survived were enrolled as survival group (5 patients). The Mann-Whitney U test was used for comparison of general data between the two groups. The label-free quantitative proteomic method was used for identification and quantitative analysis of plasma exosome proteins to screen out differentially expressed proteins, and a functional enrichment analysis was performed. R-3.5.1 software was used to perform a hierarchical cluster analysis of differentially expressed proteins to analyze the biological processes involving these proteins. Results A total of 860 proteins were identified by the exosome proteomic analysis, and according to the criteria of upregulation > 1.2 folds or downregulation > 1.2 folds (P < 0.05), there were 116 differentially expressed proteins. Compared with the liver transplantation/death group, the survival group had 62 upregulated proteins and 54 downregulated proteins. The bioinformatics analysis showed that these differentially expressed proteins mainly participated in immune reaction, signal transduction, vesicle-mediated transport, cell death, and cell proliferation and were closely associated with the signaling pathways including inflammatory response, carbohydrate and amino acid metabolism, hepatocyte injury, and hepatocyte regeneration. Conclusion Differentially expressed proteins screened out by the label-free quantitative proteomic method can be used as serological markers for the early diagnosis and prognostic evaluation of ACLF.
- Acute-On-Chronic Liver Failure /
- Exosomes /
- Proteomics /
- Computational Biology

HTML全文

图 1 差异蛋白GO功能的富集分析结果

注: CC, 细胞组分; MF, 分子功能; BP, 生物学过程; 条形图右侧为P值。

下载: 全尺寸图片幻灯片

图 2 蛋白质互作分析结果

注: 红色圈内表示上调表达蛋白, 绿圈内表示下调表达蛋白。

下载: 全尺寸图片幻灯片

图 3 KEGG信号通路富集结果

注: 颜色代表富集度[-log₁₀(P值)], 圆圈大小表示基因数目。

下载: 全尺寸图片幻灯片

表 1 液相色谱洗脱条件

时间(min)	流动相B比例(%)
0	6
8	15
60	32
79	40
80	95
85	95
86	6
90	3

下载: 导出CSV

表 2 两组患者一般资料比较

项目	生存组(n=5)	肝移植/死亡组(n=5)	Z值	P值
男性[例(%)]	3(60)	3(60)
年龄(岁)	43.80(42.00~53.00)	44.60(33.00~51.00)	0.17	0.22
ALT(U/L)	115.46(9.70~421.20)	242.58(31.30~781.90)	1.36	0.22
AST(U/L)	131.70(49.90~168.90)	228.06(50.90~496.50)	0.52	0.69
凝血酶原活动度	23.20(10.00~34.00)	31.06(15.30~48.00)	0.94	0.42
TBil(μmol/L)	360.30(213.40~498.60)	450.46(340.00~690.80)	0.52	0.69
DBil(μmol/L)	226.90(142.40~311.10)	295.70(102.30~418.70)	0.73	0.55
AFP(ng/ml)	91.20(6.25~290.10)	184.21(7.42~591.4)	0.52	0.69
WBC(×10⁹/L)	4.23(1.48~5.71)	8.50(7.40~10.43)	0.94	0.42
RBC(×10¹²/L)	3.22(2.44~3.74)	3.45(2.99~4.26)	0.31	0.84
PLT(×10⁹/L)	88.00(35.00~163.00)	85.40(42.00~121.00)	0.31	0.84
注: HLA, 人类淋巴细胞抗原。

下载: 导出CSV

表 3 (肝移植/死亡组)/生存组上调差异蛋白

基因	蛋白编号	差异蛋白	差异倍数	P值
IGHV4-4	A0A075B6R2	Ig重变4-4	747.718	0.049 58
IGHV3-21	A0A0B4J1V1	Ig重变3-21	458.288	0.043 69
IGHV3-73	A0A0B4J1V6	Ig重变3-73	450.090	0.010 41
IGKV1-8	A0A0C4DH67	Igκ变量1-8	222.641	0.001 08
IGKV1D-12	P01611	Igκ变量1D-12	181.438	0.046 98
DNM1L	G8JLD5	动力相关蛋白1(Drp1)	178.461	0.010 70
DIAPH1	H9KV28	透明基因相关成蛋白1(Diaph1)	153.400	0.041 52
NCOR1	J3KRE4	核受体共抑制因子1(NCoR1)	128.299	0.006 20
HPRT1	P00492	次黄嘌呤磷酸核糖基转移酶	113.501	0.008 19
IGLV3-19	P01714	Igλ变量3-19	108.399	0.032 01
IGHV4-39	P01824	Ig重变4-39	101.752	0.004 34
ASL	P04424	精氨琥珀酸裂解酶(ASL)	97.605	0.001 68
RPN2	P04844	核糖体结合蛋白Ⅱ	93.996	0.034 03
HLA-A	P05534	HLAⅠ类组织相容性抗原, A-24链	88.826	0.000 19
GSTP1	P09211	谷胱甘肽S-转移酶P	83.988	0.028 45

下载: 导出CSV

表 4 (肝移植/死亡组)/生存组下调差异蛋白

基因	蛋白编号	差异蛋白	差异倍数	P值
SDCBP	SDCBP	同线蛋白1	0.104 87	0.048 88
H3F3B	K7EMV3	组蛋白H3	0.092 72	0.042 38
WBP2	K7EIJ0	WW结构域结合蛋白2	0.092 49	0.042 25
DSP	P15924	桥粒斑蛋白	0.090 24	0.041 04
SLC3A2	F5GZS6	4F2细胞表面抗原重链	0.089 90	0.040 86
FTL	P02792	铁蛋白轻链	0.086 97	0.039 28
KRT6A	P02538	角蛋白, Ⅱ型细胞骨架6A	0.085 11	0.038 28
KRT2	P35908	角蛋白Ⅱ型表皮细胞骨架2	0.081 73	0.036 46
EHD4	Q9H223	EH含域蛋白4	0.081 67	0.036 42
SDCBP	G5EA09	同线蛋白, 同型CRA_a	0.081 40	0.036 29
KRT14	P02533	角蛋白, Ⅱ型细胞骨架14	0.080 83	0.035 97
F11	P03951	凝血因子Ⅺ	0.080 10	0.035 57
ANXA6	P08133	膜联蛋白A6	0.075 99	0.033 36
TFRC	P02786	转铁蛋白受体1	0.075 67	0.033 19
KPRP	Q5T749	角化细胞富含脯氨酸蛋白	0.073 74	0.032 15

下载: 导出CSV

参考文献(33)

[1]	TANG CM, YAU TO, YU J. Management of chronic hepatitis B infection: Current treatment guidelines, challenges, and new developments[J]. World J Gastroenterol, 2014, 20(20): 6262-6278. DOI: 10.3748/wjg.v20.i20.6262.
[2]	SARIN SK, KEDARISETTY CK, ABBAS Z, et al. Acute-on-chronic liver failure: Consensus recommendations of the Asian Pacific Association for the Study of the Liver (APASL) 2014[J]. Hepatol Int, 2014, 8(4): 453-471. DOI: 10.1007/s12072-014-9580-2.
[3]	SARIN SK, CHOUDHURY A. Acute-on-chronic liver failure: Terminology, mechanisms and management[J]. Nat Rev Gastroenterol Hepatol, 2016, 13(3): 131-149. DOI: 10.1038/nrgastro.2015.219.
[4]	HIRSOVA P, IBRAHIM SH, VERMA VK, et al. Extracellular vesicles in liver pathobiology: Small particles with big impact[J]. Hepatology, 2016, 64(6): 2219-2233. DOI: 10.1002/hep.28814.
[5]	XU S, ZHANG LP. Research advances in the role of exosomes in non-neoplastic liver diseases[J]. J Clin Hepatol, 2020, 36(4): 940-943. DOI: 10.3969/j.issn.1001-5256.2020.04.052. 徐帅, 张立平. 外泌体在非肿瘤性肝脏疾病中的研究进展[J]. 临床肝胆病杂志, 2020, 36(4): 940-943. DOI: 10.3969/j.issn.1001-5256.2020.04.052.
[6]	VERMA VK, LI H, WANG R, et al. Alcohol stimulates macrophage activation through caspase-dependent hepatocyte derived release of CD40L containing extracellular vesicles[J]. J Hepatol, 2016, 64(3): 651-660. DOI: 10.1016/j.jhep.2015.11.020.
[7]	NOJIMA H, FREEMAN CM, SCHUSTER RM, et al. Hepatocyte exosomes mediate liver repair and regeneration via sphingosine-1-phosphate[J]. J Hepatol, 2016, 64(1): 60-68. DOI: 10.1016/j.jhep.2015.07.030.
[8]	Liver Failure and Artificial Liver Group, Chinese Society of Infectious Diseases, Chinese Medical Association; Severe Liver Disease and Artificial Liver Group, Chinese Society of Hepatology, Chinese Medicial Association. Guideline for diagnosis and treatment of liver failure(2018)[J]. J Clin Hepatol, 2019, 35(1): 38-44. DOI: 10.3969/j.issn.1001-5256.2019.01.007. 中华医学会感染病学分会肝衰竭与人工肝学组, 中华医学会肝病学分会重型肝病与人工肝学组. 肝衰竭诊治指南(2018年版)[J]. 临床肝胆病杂志, 2019, 35(1): 38-44. DOI: 10.3969/j.issn.1001-5256.2019.01.007.
[9]	JING HM, ZHANG T, YANG F, et al. Manganese-related differential proteomic studies of cerebrospinal fluid from rats by label free method[J]. J Toxicol, 2012, 26(4): 247-253. https://www.cnki.com.cn/Article/CJFDTOTAL-WSDL201204001.htm 敬海明, 张拓, 杨帆, 等. Label free方法研究大鼠脑脊液中锰毒性相关的差异蛋白质组[J]. 毒理学杂志, 2012, 26(4): 247-253. https://www.cnki.com.cn/Article/CJFDTOTAL-WSDL201204001.htm
[10]	DEMORY BECKLER M, HIGGINBOTHAM JN, FRANKLIN JL, et al. Proteomic analysis of exosomes from mutant KRAS colon cancer cells identifies intercellular transfer of mutant KRAS[J]. Mol Cell Proteomics, 2013, 12(2): 343-355. DOI: 10.1074/mcp.M112.022806.
[11]	JI H, GREENING DW, BARNES TW, et al. Proteome profiling of exosomes derived from human primary and metastatic colorectal cancer cells reveal differential expression of key metastatic factors and signal transduction components[J]. Proteomics, 2013, 13(10-11): 1672-1686. DOI: 10.1002/pmic.201200562.
[12]	ALBILLOS A, LARIO M, ÁLVAREZ-MON M. Cirrhosis-associated immune dysfunction: Distinctive features and clinical relevance[J]. J Hepatol, 2014, 61(6): 1385-1396. DOI: 10.1016/j.jhep.2014.08.010.
[13]	GUO YY, YUAN CB, YU HY, et al. Difference of T lymphocyte subsets in peripheral blood of children with chronic hepatitis B and liver failure[J]. Chin Hepatol, 2014, 19(7): 515-517. https://www.cnki.com.cn/Article/CJFDTOTAL-ZUAN201407011.htm 郭银燕, 袁春蓓, 俞海英, 等. 慢性乙型肝炎与肝衰竭患儿外周血T淋巴细胞亚群的差异[J]. 肝脏, 2014, 19(7): 515-517. https://www.cnki.com.cn/Article/CJFDTOTAL-ZUAN201407011.htm
[14]	VEIGA-PARGA T, SEHRAWAT S, ROUSE BT. Role of regulatory T cells during virus infection[J]. Immunol Rev, 2013, 255(1): 182-196. DOI: 10.1111/imr.12085.
[15]	SMIRNOVA E, GRIPARIC L, SHURLAND DL, et al. Dynamin-related protein Drp1 is required for mitochondrial division in mammalian cells[J]. Mol Biol Cell, 2001, 12(8): 2245-2256. DOI: 10.1091/mbc.12.8.2245.
[16]	YANG X, WANG H, NI HM, et al. Inhibition of Drp1 protects against senecionine-induced mitochondria-mediated apoptosis in primary hepatocytes and in mice[J]. Redox Biol, 2017, 12: 264-273. DOI: 10.1016/j.redox.2017.02.020.
[17]	XU S, PI H, CHEN Y, et al. Cadmium induced Drp1-dependent mitochondrial fragmentation by disturbing calcium homeostasis in its hepatotoxicity[J]. Cell Death Dis, 2013, 4: e540. DOI: 10.1038/cddis.2013.7.
[18]	PALMA E, MA X, RIVA A, et al. Dynamin-1-like protein inhibition drives megamitochondria formation as an adaptive response in alcohol-induced hepatotoxicity[J]. Am J Pathol, 2019, 189(3): 580-589. DOI: 10.1016/j.ajpath.2018.11.008.
[19]	GALLOWAY CA, LEE H, BROOKES PS, et al. Decreasing mitochondrial fission alleviates hepatic steatosis in a murine model of nonalcoholic fatty liver disease[J]. Am J Physiol Gastrointest Liver Physiol, 2014, 307(6): G632-641. DOI: 10.1152/ajpgi.00182.2014.
[20]	YU T, WANG L, LEE H, et al. Decreasing mitochondrial fission prevents cholestatic liver injury[J]. J Biol Chem, 2014, 289(49): 34074-34088. DOI: 10.1074/jbc.M114.588616.
[21]	STRITT S, NURDEN P, TURRO E, et al. A gain-of-function variant in DIAPH1 causes dominant macrothrombocytopenia and hearing loss[J]. Blood, 2016, 127(23): 2903-2914. DOI: 10.1182/blood-2015-10-675629.
[22]	LYNCH ED, LEE MK, MORROW JE, et al. Nonsyndromic deafness DFNA1 associated with mutation of a human homolog of the Drosophila gene diaphanous[J]. Science, 1997, 278(5341): 1315-1318. DOI: 10.1126/science.278.5341.1315
[23]	YANG J, ZHOU L, ZHANG Y, et al. DIAPH1 Is Upregulated and inhibits cell apoptosis through ATR/p53/Caspase-3 signaling pathway in laryngeal squamous cell carcinoma[J]. Dis Markers, 2019, 2019: 6716472. DOI: 10.1155/2019/6716472.
[24]	LIU D, FU X, WANG Y, et al. Protein diaphanous homolog 1 (Diaph1) promotes myofibroblastic activation of hepatic stellate cells by regulating Rab5a activity and TGFβ receptor endocytosis[J]. FASEB J, 2020, 34(6): 7345-7359. DOI: 10.1096/fj.201903033R.
[25]	SUN Z, FENG D, FANG B, et al. Deacetylase-independent function of HDAC3 in transcription and metabolism requires nuclear receptor corepressor[J]. Mol Cell, 2013, 52(6): 769-782. DOI: 10.1016/j.molcel.2013.10.022.
[26]	BURRAGE LC, MADAN S, LI X, et al. Chronic liver disease and impaired hepatic glycogen metabolism in argininosuccinate lyase deficiency[J]. JCI Insight, 2020, 5(4). DOI: 10.1172/jci.insight.132342.
[27]	UCHIHARA T, MIYAKE K, YONEMURA A, et al. Extracellular vesicles from cancer-associated fibroblasts containing annexin A6 induces FAK-YAP activation by stabilizing β1 integrin, enhancing drug resistance[J]. Cancer Res, 2020, 80(16): 3222-3235. DOI: 10.1158/0008-5472.CAN-19-3803.
[28]	ALVAREZ-GUAITA A, BLANCO-MUÑOZ P, MENESES-SALAS E, et al. Annexin A6 is critical to maintain glucose homeostasis and survival during liver regeneration in mice[J]. Hepatology, 2020, 72(6): 2149-2164. DOI: 10.1002/hep.31232.
[29]	TORTI SV, TORTI FM. Iron and cancer: More ore to be mined[J]. Nat Rev Cancer, 2013, 13(5): 342-355. DOI: 10.1038/nrc3495.
[30]	ALKHATEEB AA, CONNOR JR. The significance of ferritin in cancer: Anti-oxidation, inflammation and tumorigenesis[J]. Biochim Biophys Acta, 2013, 1836(2): 245-254. DOI: 10.1016/j.bbcan.2013.07.002.
[31]	LUNOVA M, GOEHRING C, KUSCUOGLU D, et al. Hepcidin knockout mice fed with iron-rich diet develop chronic liver injury and liver fibrosis due to lysosomal iron overload[J]. J Hepatol, 2014, 61(3): 633-641. DOI: 10.1016/j.jhep.2014.04.034.
[32]	WANG AR, LI XL, MEN QS, et al. Serum ferritin and liver disease[J/CD]. Chin J Liver Dis(Electronic Edition), 2020, 12(4): 34-37. DOI: 10.3969/j.issn.1674-7380.2020.04.006. 王傲然, 李晓玲, 门秋爽, 等. 血清铁蛋白与肝脏疾病研究进展[J/CD]. 中国肝脏病杂志(电子版), 2020, 12(4): 34-37. DOI: 10.3969/j.issn.1674-7380.2020.04.006.
[33]	DENG JH, CHEN ZP, HUANG MM, et al. Clinical significance of Ferritin Light Chain and its expression in serum of patients with chronic hepatopathy[J]. Modern Prevent Med, 2016, 43(18): 3444-3447. https://www.cnki.com.cn/Article/CJFDTOTAL-XDYF201618046.htm 邓敬桓, 陈智平, 黄美梦, 等. FTL在慢性肝病患者血清中的表达及临床意义[J]. 现代预防医学, 2016, 43(18): 3444-3447. https://www.cnki.com.cn/Article/CJFDTOTAL-XDYF201618046.htm