腹膜透析滤出液外泌体miR-200a在不同腹膜转运特性患者中的表达差异及其生物学功能预测

doi:10.3969/j.issn.1674-8115.2021.01.007

摘要/Abstract

摘要：

目的·提取和鉴定腹膜透析滤出液（peritoneal dialysis effluent，PDE）外泌体，比较不同腹膜转运特性患者PDE外泌体miR-200a的表达水平，预测其潜在靶基因及参与的生物学过程。方法·将20例腹膜透析（peritoneal dialysis，PD）患者按照腹膜平衡实验（peritoneal equilibrium test，PET）值分为H组（高转运/高平均转运）和L组（低转运/低平均转运）。每位患者收集500 mL留腹过夜PDE，浓缩后超速离心，透射电子显微镜（transmission electron microscope，TEM）、纳米粒子跟踪分析（nanoparticle tracking analysis，NTA）和Western blotting鉴定外泌体。提取外泌体miRNA，实时荧光定量PCR检测2组患者PDE外泌体miR-200a的表达水平，分析其与PET值和超滤量的相关性。利用Targetscan、miRmap和miRWalk数据库预测miR-200a的靶基因，用DAVID数据库进行基因功能（gene ontology，GO）富集分析。结果·浓缩的PDE超速离心后提取到的外泌体，TEM下呈双层膜包裹的圆形囊泡，NTA显示其直径大多在50~150 nm，表面具有外泌体标志蛋白CD9和CD63。L组中PDE外泌体miR-200a的相对表达量显著高于H组，并且与PET值呈负相关（r=-0.871，P=0.000），与4 h超滤量呈正相关（r=0.448，P=0.048），但与24 h超滤量无相关性（r=0.355，P=0.125）。生物信息学分析发现共有679种基因可由3个数据库共同预测到；GO分析显示这些靶基因不仅参与正向调控间充质细胞增殖，也在金属离子结合方面有一定富集。结论·PDE中存在外泌体，腹膜转运特性较低的患者PDE外泌体miR-200a相对表达量较高，其与PET值呈负相关，与4 h超滤量呈正相关。miR-200a的潜在靶基因众多，可能通过不同生物学过程影响腹膜转运特性。

关键词: 腹膜透析滤出液, 外泌体, miR-200a, 腹膜转运特性, 生物信息学

Abstract:

Objective·To extract and identify the exosomes in peritoneal dialysis effluent (PDE), compare the different expression levels of PDE exosomal miR-200a in patients with different peritoneal transport characteristics, predict the potential target genes of miR-200a and analyze the related biological processes.

Methods·Twenty stable peritoneal dialysis (PD) patients were divided into two groups, i.e., H group (high/high average) and L group (low/low average) according to the values of peritoneal equilibration test (PET). Overnight PDE 500 mL from each patient was collected, concentrated, and ultracentrifuged to obtain exosomes. Transmission electron microscope (TEM), nanoparticle tracking analysis (NTA) and Western blotting were used to identify and characterize the exosomes. Exosomal miRNAs were extracted and the expression levels of PDE exosomal miR-200a in the two groups were determined by real-time quantitative PCR. The relations between exosomal miR-200a levels and PET values or ultrafiltration volume (UF) were analyzed. The target genes of miR-200a were predicted by Targetscan, miRmap and miRWalk, and DAVID was used to perform gene ontology (GO) enrichment analysis.

Results·The exosomes extracted from concentrated PDE by ultracentrifugation exhibited a round and bilayer membrane-enveloped vesicle structure under TEM with the diameters ranging from 50-150 nm. The exosomes also expressed the particular molecular marker CD9 and CD63. The relative expression level of PDE exosomal miR-200a in L group was higher than that of H group. The expression levels were negatively correlated with PET values (r=-0.871, P=0.000) and positively correlated with 4 h UF (r=0.448, P=0.048). But there was no relationship between miR-200a and 24 h UF (r=0.355, P=0.125). Bioinformatic results showed that there were 679 target genes of miR-200a that could be predicted by all the three databases. GO analysis suggested that these genes not only participated in positive regulation of mesenchymal cells proliferation, but also focused on ion binding, especially metal cation binding.

Conclusion·Exosomes indeed exist in PDE. The relative expression level of exosomal miR-200a is higher in the PDE from the patients with low peritoneal dialysis transport characteristics. The relative quantity of miR-200a is negatively correlated with the values of PET and positively correlated with 4 h UF. There are abundant potential target genes of miR-200a, indicating that it may affect the peritoneal transport characteristics by different biological processes.

Key words: peritoneal dialysis effluent, exosome, miR-200a, peritoneal dialysis characteristics, bioinformatics

中图分类号:

R459.5

佟琰, 方均燕, 邓海, 宋阿会, 李璞, 刘英莉. 腹膜透析滤出液外泌体miR-200a在不同腹膜转运特性患者中的表达差异及其生物学功能预测[J]. 上海交通大学学报(医学版), 2021, 41(1): 42-48.

Yan TONG, Jun-yan FANG, Hai DENG, A-hui SONG, Pu LI, Ying-li LIU. Different expression levels of exosomal miR-200a in peritoneal dialysis effluent from patients with different peritoneal transport characteristics and prediction of its biological function[J]. JOURNAL OF SHANGHAI JIAOTONG UNIVERSITY (MEDICAL SCIENCE), 2021, 41(1): 42-48.

图/表 6

图1 PDE外泌体的鉴定Note:A. Bilayer vesicle structure of exosomes under TEM. B. The diameters of exosomes were 50-150 nm detected by NTA. C. CD9 and CD63 expression detected by Western blotting.

Fig 1 Identification of PDE exosomes

表1 PD患者的临床资料

Tab 1 Clinical characteristics of PD patients

Item	H group （n=10）	L group （n=10）	t/U value	P value
Gender （male/female）/n	4/6	5/5	-	0.999
Age/year	61.90±14.29	61.00±8.72	0.170	0.867
Dialysis time/month	34.60±20.43	50.70±23.39	1.640	0.119
PET value	0.75±0.06	0.59±0.04	6.631	0.000
eGFR/[mL·（min·1.73 m²）^-1]	2.50 （0, 5.67）	0 （0, 4.54）	44	0.660
Kt/V/（mL·min^-1）	2.41 （1.72, 2.69）	1.79 （1.75, 1.85）	36	0.315

图2 2组PDE外泌体miR-200a表达水平的比较

Fig 2 Comparison of expression of PDE exosomal miR-200a between the two groups

图3 PDE外泌体miR-200a与PET值、4 h和24 h超滤量的相关性分析

Fig 3 Correlation analysis between PDE exosomal miR-200a and PET values, 4 h and 24 h ultrafiltration volume

图4 miR-200a潜在靶基因预测及EMT相关靶基因表达蛋白的PPI模块Note:A. Venn diagram of miR-200a target genes. B. PPI modules of EMT-related target genes expressing proteins. The yellow nodes are the proteins of target genes, and the blue nodes are the proteins interacted with them.

Fig 4 Prediction of target genes of miR-200a and PPI modules of EMT-related target genes expressing proteins

图5 miR-200a靶基因的GO富集分析Note:The ordinate represents the enriched items, and the abscissa represents the negative logarithm of P values.

Fig 5 GO enrichment analysis of miR-200a target genes

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