Analysis of miRNA expression in Echinococcus granulosus protoscoleces isolated from sheep liver and lung

doi:10.12140/j.issn.1000-7423.2019.02.004

Abstract

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

CLC Number:

R383.33

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