绵羊肝脏、肺脏细粒棘球蚴原头节miRNA表达特征分析

doi:10.12140/j.issn.1000-7423.2019.02.004

摘要/Abstract

摘要：

目的分析绵羊肝脏及肺脏分离的细粒棘球蚴原头节（HPSC、LPSC）的微小RNA（miRNA）表达情况,筛选出寄生部位特异性miRNA,为后续miRNA生物信息学分析提供依据。方法从西宁市郊屠宰场采集带有包囊的新鲜绵羊肝脏、肺脏,经分离、过滤、沉淀获得棘球蚴原头节。用TRIzol法提取原头节总RNA,构建HPSC、LPSC小片段RNA(sRNA)文库,采用高通量测序技术进行深度测序,将测序获得的原始数据与miRBase、Pre-miRNA、RFam、Repbase等4个公共数据库进行比对,获得已知miRNA的注释信息。利用mfold软件预测未比对上任何注释信息的miRNA的二级结构。比较HPSC、LPSC中miRNA表达量的差异。结果 HPSC和LPSC文库分别获得10 182 508条和11 133 735条有效序列,大部分序列长18~22 nt。其中22 nt的序列最多,分别占20.9%（2 128 728/10 182 508）和24.6%（2 737 570/11 133 735）;19 nt及21 nt次之,分别占14.0%（1 429 846/10 182 508）、18.5%（1 880 607/10 182 508）和16.9%（1 875 698/11 133 735）、16.9%（1 885 560/11 133 735）。在HPSC和LPSC文库中,细粒棘球蚴原头节已知miRNA分别有87条和79条,新miRNA分别占341条和339条,与其他寄生虫（日本血吸虫、曼氏血吸虫、扁虫及三代虫等）保守的miRNA分别有58条和62条。在细粒棘球蚴原头节已知miRNA中,除egr-miR-36a-3p、egr-miR-36b-3p、egr-miR-10229a-5p、egr-miR-10233-5p、egr-miR-10240-3p、egr-miR-10243-5p、egr-mir-10252-5p、egr-mir-10287-5p等8个miRNA在HPSC中特异表达外,其余miRNA在2个文库均表达且表达量一致;在与其他寄生虫保守的miRNA中,有50条在HPSC和LPSC中均表达,有8条和12条分别在2个文库中特异表达。在新的miRNA中,在HPSC和LPSC文库特异表达的分别为78和76条。HPSC和LPSC文库共筛选出miRNA 574条,其中392条miRNA在2个文库中均表达,表达量居前10位的为egr-miR-1-5p、egr-miR-2a-3p、egr-miR-2c-3p、egr-miR-2b-3p、egr-miR-7-5p、egr-let-7-5p、egr-miR-7b-5p、egr-miR-9-5p、egr-miR-9-3p、egr-miR-10a-5p。LPSC与HPSC文库相比较,LPSC有124条miRNA上调2倍以上,30条miRNA下调0.5倍以下。在筛选出的miRNA中,HPSC和LPSC中分别有94条和88条特异表达。结论本研究发现HPSC及LPSC中存在特异表达miRNA。

关键词: 细粒棘球绦虫, miRNA, 表达特征分析

Abstract:

Objective The expression of microRNA (miRNA) in Echinococcus granulosus protoscoleces isolated from liver (hepatic protoscoleces, HPSC) and lung (lung protoscoleces, LPSC) of infected sheep was analyzed, and the specific miRNAs expressed in E. granulosus were identified by screening available databases in order to provide E. granulosus miRNA information for further study. Methods The fresh sheep livers and lungs with hydatid cysts were collected from a slaughter house near the suburb of Xining City. The hydatid cysts were extracted, filtered and precipitated to obtain pure protoscoleces of E. granulosus. The total RNA was extracted from cysts using TRIzol, and the HPSC and LPSC small RNA (sRNA) libraries were constructed. The deep sequencing was performed on these libraries using high-throughput sequencing technology. The obtained sequences were used to compare with databases including miRBase, Pre-miRNA database, RFam and Repbase to obtain E. granulosus specific miRNA information. The secondary structure of E. granulosus miRNA was predicted using mfold software. The differences in miRNA expression between HPSC and LPSC were compared. Results The E. granulosus HPSC and LPSC sRNA libraries were successfully constructed and total 10 182 508 and 11 133 735 effective sequences, respectively, were obtained. Most of the sequences were 18-22 nucleotide (nt) long, of which 22 nt had the highest frequency accounting for 20.9% (2 128 728/10 182 508) for HPSC and 24.6%(2 737 570/11 133 735) for LPSC, followed by 19 nt (14.0%, 1 429 846/10 182 508; 18.5%, 1 880 607/10 182 508) and 21 nt (16.9%, 1 875 698/11 133 735; 16.9%, 1 885 560/11 133 735), respectively. Except for 87 and 79 known miRNA, total 341 and 339 new miRNAs were identified in the constructed E. granulosus HPSC and LPSC sRNA libraries, respectively. Among them 58 for HPSC and 62 for LPSC were conserved with sequences of Schistosoma japonicum, S. mansoni, Platyhelminthes and Gyrodactylus. In the known miRNA of E. granulosus, 8 miRNA including egr-miR-36a-3p, egr-miR-36b-3p, egr-miR-10229a-5p, egr-miR-10233-5p, egr-miR-10240-3p, egr-miR-10243-5p, egr-mir-10252-5p and egr-mir-10287-5p were specifically expressed in the HPSC library, and the remaining miRNA were expressed in both HPSC and LPSC sRNA libraries at the same level. Among other helminth-conserved miRNA, 50 were co-expressed in both HPSC and LPSC, 8 were specifically expressed in HPSC and 12 in LPSC. Among the new identified miRNA, 78 were specifically expressed in HPSC and 76 in LPSC. Of the 574 miRNA identified in two libraries, 392 miRNA were co-expressed in both libraries with following top 10 highest expression miRNAs: egr-miR-1-5p, egr-miR-2a-3p, egr-miR-2c-3p, egr-miR-2b-3p, egr-miR-7-5p, egr-let-7-5p, egr-miR-7b-5p, egr-miR-9-5p, egr-miR-9-3p, egr-miR-10a-5p. Compared with HPSC, 124 miRNAs were up-regulated by more than 2-fold and 30 miRNAs down-regulated by 0.5-fold in LPSC. Among the total miRNA identified in both libraries, 94 and 88 were specifically expressed in HPSC and LPSC, respectively. Conclusion This study identified the specific expression of miRNA in HPSC and LPSC.

Key words: Echinococcus granulosus, miRNA, Expression analysis

中图分类号:

R383.33

付世强, 樊海宁, 王海久, 周瀛, 曹得萍, 李衍飞, 王志鑫, 任利. 绵羊肝脏、肺脏细粒棘球蚴原头节miRNA表达特征分析[J]. 中国寄生虫学与寄生虫病杂志, 2019, 37(2): 137-143.

Shi-qiang FU, Hai-ning FAN, Hai-jiu WANG, Ying ZHOU, De-ping CAO, Yan-fei LI, Zhi-xin WANG, Li REN. Analysis of miRNA expression in Echinococcus granulosus protoscoleces isolated from sheep liver and lung[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2019, 37(2): 137-143.

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miRNA名称 miRNA name	相对表达量Relative expression level
miRNA名称 miRNA name	HPSC	LPSC
egr-miR-1-5p	4 749.9	7 137.9
egr-miR-2a-3p	6 293.2	3 235.5
egr-miR-2c-3p	11 388.6	7 617.0
egr-miR-2b-3p	14 499.0	8 344.5
egr-miR-7-5p	2 194.3	4 180.1
egr-let-7-5p	29 471.2	45 604.0
egr-miR-7b-5p	1 034.5	2 420.5
egr-miR-9-5p	5 854.9	8 052.8
egr-miR-9-3p	560.3	271.7
egr-miR-10a-5p	10 095.7	6 486.7
egr-miR-61-3p	2 920.6	2 265.7
egr-miR-71-5p	68 100.4	112 588.0
egr-miR-87-3p	302.5	519.4
egr-miR-96-5p	3 578.9	2 454.4
egr-miR-124b-3p	5 269.5	3 838.5
egr-miR-125-5p	20 687.7	14 484.1
egr-miR-184-3p	16 770.3	19 853.5
egr-miR-190-5p	681.9	1 620.3
egr-miR-219-5p	3 884.7	2 596.1
egr-miR-277a-3p	3 373.9	3 015.3
egr-miR-281-3p	3 787.8	2 752.1
egr-miR-745-3p	5 386.4	5 501.8
egr-miR-2162-3p	3 603.1	1 532.2
egr-miR-3479a-3p	2 825.3	1 730.9
egr-miR-4989-3p	14 009.5	8 662.5
sma-miR-1b-3p	1 961.5	3 262.4
gsa-miR-87-3p	6 331.6	12 628.4
sme-mir-749-p3	4.4	8.5
bmo-mir-6497-p5	1.7	5.6
lgi-miR-100	1.4	98.4
prd-let-7-5p	5.0	509.4

miRNA	差异倍数Fold change	上调/下调Up/down
prd-let-7-5p	101.4	上调Up
lgi-miR-100	71.8	上调Up
aga-miR-92a	46.3	上调Up
aae-miR-92a-3p	46.3	上调Up
tur-miR-92-3p	46.3	上调Up
PC-3p-34218_161	16.6	上调Up
PC-3p-95756_44	11.9	上调Up
PC-3p-48670_108	10.2	上调Up
PC-3p-52795_98	8.8	上调Up
PC-3p-125187_29	8.3	上调Up
PC-3p-70159_69	7.5	上调Up
PC-3p-71169_67	7.3	上调Up
PC-3p-68407_71	6.8	上调Up
PC-5p-102730_40	6.8	上调Up
PC-3p-90370_48	6.8	上调Up
PC-3p-97537_43	6.8	上调Up
PC-5p-68303_71	6.6	上调Up
PC-5p-29352_189	6.5	上调Up
PC-3p-22591_246	6.3	上调Up
PC-3p-126082_29	6.3	上调Up
PC-3p-27703_201	6.1	上调Up
PC-3p-47625_111	6.0	上调Up
PC-5p-105060_39	6.0	上调Up
PC-3p-37448_146	5.8	上调Up
PC-3p-22154_250	5.4	上调Up
PC-3p-56267_91	5.4	上调Up
PC-3p-56802_90	5.2	上调Up
PC-5p-63015_79	5.1	上调Up
egr-miR-8-3p	0.2	下调Down
egr-miR-96-3p	0.2	下调Down
PC-5p-49121_107	0.2	下调Down