Identification of miRNAs in the infectious third stage larvae and parasitic female adult of Strongyloides stercoralis

doi:10.12140/j.issn.1000-7423.2023.04.003

Abstract

Abstract:

Objective To identify the microRNA (miRNA) of the Strongyloides stercoralis infective third-stage larvae (iL₃) and parasitic female adult (pF), and analyze the differential expression of miRNA and the function of their target genes. Methods The iL₃ was collected from the feces of dogs infected with S. stercoralis. Healthy dogs were subcutaneously injected with 4 000-5 000 iL₃ at the back of the neck. The pF was collected from the dog intestine 21 days post-infection. The total RNA of iL₃ and pF was extracted and sequenced. The sequencing data was aligned with the S. stercoralis genome and S. ratti miRNA to identify the conserved miRNA. The true miRNA sequences were screened out using miRDeep2 through loop structure analysis and mature sequence readings alignment. The miRNA expression profiles of iL₃ and pF were analyzed to screen the differentially expressed miRNA, and predict their target genes. Functional analysis was performed to those of unannotated miRNAs target genes with the values of log₂(Fold chang) > 1 and transcripts per million (TPM) > 1 000. GO enrichment analysis of differentially expressed miRNA target genes were analyzed using ClusterProfiler. Eleven unannotated or conserved differentially expressed miRNAs were selected for real-time fluorescent quantitative PCR (qRT-PCR) verification. The relative transcription level was calculated with the glyceraldehyde-3-phosphate dehydrogenase gene of S. stercoralis (GenBank accession number: BI773092.1) as reference gene. Results A total of 265 miRNA were identified, including 130 conserved miRNAs and 135 novel miRNAs, of them 134 were differentially expressed between iL₃ and pF (77 conserved and 57 unannotated miRNAs), 251 miRNAs were shared by iL₃ and pF, 10 were specific in iL₃ and 4 were specific in pF. The prediction results of differentially expressed miRNA target genes showed that of the 57 unannotated miRNA, 12 miRNA with fold chang > 2 and TPM > 1 000 have 248 target genes homologous with Caenorhabditis elegans. 35.08% (87/248) of the target genes were related to development, while the rest were related to stress, exercise and molting. Most of the highly expressed unannotated miRNA in pF target SSTP_0000114700. GO analysis of differentially expressed miRNA target genes showed that a total of 5 595 target genes were enriched, among which the top 5 enriched components were membrane components (1 821), nucleic acid binding (561), nucleus (450), proteolysis (365) and signal transduction (284). The qRT-PCR analysis showed that the log₂(fold change) of sst-miR-86-5p, sst-84-5p, sst-novel-104, sst-miR-92-3p, sst-miR-34a-3p, sst-miR-81a-5p, sst-miR-1-3p, sst-novel-108, sst-miR-124-5p, sst-miR-50-3p, sst-novel-51 were -2.12, 6.39, 4.46, -3.69, -3.69, 2.34, -2.48, -2.41, -2.30, 2.25, -3.32; and the log₂(fold change) obtained from transcriptome analysis were -3.05, 4.98, 4.07, -4.9, -3.66, 0.98, -3.79, -2.61, -0.99, 0.63, -1.55, respectively. The upregulation and downregulation trends obtained from qRT-PCR and transcriptome analysis are consistent. Conclusion The miRNA expression profiles and differentially expressed miRNAs of S. stercoralis infective third-stage larvae and parasitic female adult are identified. The differentially expressed miRNAs are associated with the growth, development, and reproductive function of S. stercoralis.

Key words: Strongyloides stercoralis, Infective third-stage larvae, Parasitic female adult, miRNA

CLC Number:

R383.19

QIN Peixi, ZHOU Caixian, LU Zhigang, ZHANG Biying, ZHOU Taoxun, HU Min. Identification of miRNAs in the infectious third stage larvae and parasitic female adult of Strongyloides stercoralis[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2023, 41(4): 412-420.

Figures/Tables 9

Table 1

Table 2

Fig. 1

Fig. 2

Fig. 3

Table 3

Fig. 4

Fig. 5

Fig. 6

References 36

[1]	Beknazarova M, Whiley H, Ross K. Strongyloidiasis: a disease of socioeconomic disadvantage[J]. Int J Environ Res Public Health, 2016, 13(5): 517. doi: 10.3390/ijerph13050517
[2]	Olsen A, van Lieshout L, Marti H, et al. Strongyloidiasis: the most neglected of the neglected tropical diseases?[J]. Trans R Soc Trop Med Hyg, 2009, 103(10): 967-972. doi: 10.1016/j.trstmh.2009.02.013
[3]	Puthiyakunnon S, Boddu S, Li YJ, et al. Strongyloidiasis: an insight into its global prevalence and management[J]. PLoS Negl Trop Dis, 2014, 8(8): e3018. doi: 10.1371/journal.pntd.0003018
[4]	Hu JY. The establishment of Strongyloides stercoralis infected geril model and initial attempt to establish CRISPR/Cas9 knockout method[D]. Wuhan: Huazhong Agricultural University, 2018: 2. (in Chinese)
	(胡锦阳. 粪类圆线虫感染沙鼠模型的建立及CRISPR/Cas9基因敲除方法的初步尝试[D]. 武汉: 华中农业大学, 2018: 2.)
[5]	Marcos LA, Terashima A, Dupont HL, et al. Strongyloides hyperinfection syndrome: an emerging global infectious disease[J]. Trans R Soc Trop Med Hyg, 2008, 102(4): 314-318. doi: 10.1016/j.trstmh.2008.01.020
[6]	Starr MC, Montgomery SP. Soil-transmitted helminthiasis in the United States: a systematic review: 1940—2010[J]. Am J Trop Med Hyg, 2011, 85(4): 680-684. doi: 10.4269/ajtmh.2011.11-0214
[7]	Vasquez-Rios G, Pineda-Reyes R, Pineda-Reyes J, et al. Strongyloides stercoralis hyperinfection syndrome: a deeper understanding of a neglected disease[J]. J Parasit Dis, 2019, 43(2): 167-175. doi: 10.1007/s12639-019-01090-x
[8]	Hammond SM. An overview of microRNAs[J]. Adv Drug Deliv Rev, 2015, 87: 3-14. doi: 10.1016/j.addr.2015.05.001
[9]	Lee RC, Feinbaum RL, Ambros V. The C. elegans heterochronic gene Lin-4 encodes small RNAs with antisense complementarity to Lin-14[J]. Cell, 1993, 75(5): 843-854. doi: 10.1016/0092-8674(93)90529-y pmid: 8252621
[10]	Pasquini G, Kunej T. A map of the microRNA regulatory networks identified by experimentally validated microRNA-target interactions in five domestic animals: cattle, pig, sheep, dog, and chicken[J]. OMICS, 2019, 23(9): 448-456. doi: 10.1089/omi.2019.0082 pmid: 31381467
[11]	Song XW, Li Y, Cao XF, et al. microRNAs and their regulatory roles in plant-environment interactions[J]. Annu Rev Plant Biol, 2019, 70: 489-525 doi: 10.1146/annurev-arplant-050718-100334 pmid: 30848930
[12]	Ulusan Bağcı Ö, Caner A. The role of microRNAs in parasitology[J]. Turkiye Parazitol Derg, 2020, 44(2): 102-108. doi: 10.4274/tpd.galenos.2020.6776 pmid: 32482043
[13]	Ahmed R, Chang ZS, Younis AE, et al. Conserved miRNAs are candidate post-transcriptional regulators of developmental arrest in free-living and parasitic nematodes[J]. Genome Biol Evol, 2013, 5(7): 1246-1260. doi: 10.1093/gbe/evt086 pmid: 23729632
[14]	Ma GX, Luo YF, Zhu HH, et al. microRNAs of Toxocara canis and their predicted functional roles[J]. Parasit Vectors, 2016, 9: 229. doi: 10.1186/s13071-016-1508-3
[15]	Winter AD, Weir W, Hunt M, et al. Diversity in parasitic nematode genomes: the microRNAs of Brugia pahangi and Haemonchus contortus are largely novel[J]. BMC Genomics, 2012, 13: 4. doi: 10.1186/1471-2164-13-4 pmid: 22216965
[16]	Xu MJ, Fu JH, Nisbet AJ, et al. Comparative profiling of microRNAs in male and female adults of Ascaris suum[J]. Parasitol Res, 2013, 112(3): 1189-1195. doi: 10.1007/s00436-012-3250-x
[17]	Pomari E, Malerba G, Veschetti L, et al. Identification of miRNAs of Strongyloides stercoralis L₁ and iL₃ larvae isolated from human stool[J]. Sci Rep, 2022, 12(1): 9957. doi: 10.1038/s41598-022-14185-y pmid: 35705621
[18]	Zhang Y. Genome-wide identfication and characterization of novel LncRNAs and extracellular vesicle preliminary study in Strongyloides stercoralis[D]. Wuhan: Huazhong Agricultural University, 2019: 17. (in Chinese)
	(张映. 粪类圆线虫lncRNA的鉴定和验证以及细胞外囊泡的初步研究[D]. 武汉: 华中农业大学, 2019: 17.)
[19]	Langmead B, Trapnell C, Pop M, et al. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome[J]. Genome Biol, 2009, 10(3): R25. doi: 10.1186/gb-2009-10-3-r25
[20]	Robinson MD, McCarthy DJ, Smyth GK. edgeR: a bioconductor package for differential expression analysis of digital gene expression data[J]. Bioinformatics, 2010, 26(1): 139-140. doi: 10.1093/bioinformatics/btp616 pmid: 19910308
[21]	Hunt VL, Tsai IJ, Coghlan A, et al. The genomic basis of parasitism in the Strongyloides clade of nematodes[J]. Nat Genet, 2016, 48(3):
[22]	Britton C, Laing R, Devaney E. Small RNAs in parasitic nematodes-forms and functions[J]. Parasitology, 2020, 147(8): 855-864. doi: 10.1017/S0031182019001689
[23]	Liu N, Landreh M, Cao KJ, et al. The microRNA miR-34 modulates ageing and neurodegeneration in Drosophila[J]. Nature, 2012, 482(7386): 519-523. doi: 10.1038/nature10810
[24]	Yang JR, Chen DP, He YN, et al. miR-34 modulates Caenorhabditis elegans lifespan via repressing the autophagy gene atg9[J]. Age, 2013, 35(1): 11-22. doi: 10.1007/s11357-011-9324-3
[25]	Isik M, Blackwell TK, Berezikov E. microRNA mir-34 provides robustness to environmental stress response via the DAF-16 network in C. elegans[J]. Sci Rep, 2016, 6: 36766. doi: 10.1038/srep36766
[26]	Boulias K, Horvitz HR. The C. elegans microRNA mir-71 acts in neurons to promote germline-mediated longevity through regulation of DAF-16/FOXO[J]. Cell Metab, 2012, 15(4): 439-450. doi: 10.1016/j.cmet.2012.02.014
[27]	Zhang XC, Zabinsky R, Teng YD, et al. microRNAs play critical roles in the survival and recovery of Caenorhabditis elegans from starvation-induced L1 diapause[J]. Proc Natl Acad Sci USA, 2011, 108(44): 17997-18002. doi: 10.1073/pnas.1105982108
[28]	Pérez MG, Spiliotis M, Rego N, et al. Deciphering the role of miR-71 in Echinococcus multilocularis early development in vitro[J]. PLoS Negl Trop Dis, 2019, 13(12): e0007932. doi: 10.1371/journal.pntd.0007932
[29]	Zheng YD, Guo XL, He W, et al. Effects of Echinococcus multilocularis miR-71 mimics on murine macrophage RAW264.7 cells[J]. Int Immunopharmacol, 2016, 34: 259-262. doi: 10.1016/j.intimp.2016.03.015
[30]	Yang ML, Wang YL, Jiang F, et al. miR-71 and miR-263 jointly regulate target genes chitin synthase and chitinase to control locust molting[J]. PLoS Genet, 2016, 12(8): e1006257. doi: 10.1371/journal.pgen.1006257
[31]	Davis MW, Birnie AJ, Chan AC, et al. A conserved metalloprotease mediates ecdysis in Caenorhabditis elegans[J]. Development, 2004, 131(23): 6001-6008. doi: 10.1242/dev.01454
[32]	Gamble HR, Purcell JP, Fetterer RH. Purification of a 44 kilodalton protease which mediates the ecdysis of infective Haemonchus contortus larvae[J]. Mol Biochem Parasitol, 1989, 33(1): 49-58. doi: 10.1016/0166-6851(89)90041-8
[33]	Stepek G, McCormack G, Birnie AJ, et al. The astacin metalloprotease moulting enzyme NAS-36 is required for normal cuticle ecdysis in free-living and parasitic nematodes[J]. Parasitology, 2011, 138(2): 237-248. doi: 10.1017/S0031182010001113 pmid: 20800010
[34]	Audhya A, Desai A, Oegema K. A role for Rab5 in structuring the endoplasmic reticulum[J]. J Cell Biol, 2007, 178(1): 43-56. pmid: 17591921
[35]	Sann SB, Crane MM, Lu H, et al. Rabx-5 regulates RAB-5 early endosomal compartments and synaptic vesicles in C. elegans[J]. PLoS One, 2012, 7(6): e37930. doi: 10.1371/journal.pone.0037930
[36]	Kamikura DM, Cooper JA. Clathrin interaction and subcellular localization of Ce-DAB-1, an adaptor for protein secretion in Caenorhabditis elegans[J]. Traffic, 2006, 7(3): 324-336. pmid: 16497226

引物名称 Primer name	引物序列（5'→3'） Primer sequence (5'→3')
sst-miR-86-5p-F	GCCGTAAGTGAATGCTTTGCCACAG
sst-miR-86-5p-loop	GTCGTATCCAGTGCAGGGTCCGAGGTAT-
sst-miR-86-5p-loop	TCGCACTGGATACGACAGACTG
sst-miR-84-5p-F	CGGGCTGAGGTAGTGTTAAATATTG
sst-miR-84-5p-loop	GTCGTATCCAGTGCAGGGTCCGAGGTAT-
sst-miR-84-5p-loop	TCGCACTGGATACGACAACAAT
sst-novel-104-F	CGGGCCTGAGGTAGTGTTAAATATTG
sst-novel-104-loop	GTCGTATCCAGTGCAGGGTCCGAGGTAT-
sst-novel-104-loop	TCGCACTGGATACGACAACAAT
sst-miR-92-3p-F	CGTATTGCACACGTCCCG
sst-miR-92-3p-loop	GTCGTATCCAGTGCAGGGTCCGAGGTAT-
sst-miR-92-3p-loop	TCGCACTGGATACGACTCAGGC
sst-miR-34a-3p-F	GCGCGACAGCTCACTCAACT
sst-miR-34a-3p-loop	GTCGTATCCAGTGCAGGGTCCGAGGTAT-
TCGCACTGGATACGACCTTGGC
sst-miR-81a-5P-F	CGGGCATGGGTCTATATGATTCTC
sst-miR-81a-5P-loop	GTCGTATCCAGTGCAGGGTCCGAGGTAT-
sst-miR-81a-5P-loop	TCGCACTGGATACGACCATGAG
sst-miR-1-3p-F	CGCCCGTGGAATGTAAAGAAG
sst-miR-1-3p-loop	GTCGTATCCAGTGCAGGGTCCGAGGTAT-
sst-miR-1-3p-loop	TCGCACTGGATACGACTGCATA
sst-novel-108-F	TTTGCGACCGAATCCAGGC
sst-novel-108-loop	GTCGTATCCAGTGCAGGGTCCGAGGTAT-
sst-novel-108-loop	TCGCACTGGATACGACTGTGGC
sst-miR-124-5p-F	GCGCTTTCATCCGTGACTTTAG
sst-miR-124-5p-loop	GTCGTATCCAGTGCAGGGTCCGAGGTAT-
sst-miR-124-5p-loop	TCGCACTGGATACGACCGACCT
sst-miR-50-3p-F	CGGCCCGCGAGTAATATTAGACATATCG
sst-miR-50-3p-loop	GTCGTATCCAGTGCAGGGTCCGAGGTAT-
sst-miR-50-3p-loop	TCGCACTGGATACGACCTCGAT
sst-novel-51-F	TTAAGGCACGCGGTGAATG
sst-novel-51-loop	GTCGTATCCAGTGCAGGGTCCGAGGTAT-
sst-novel-51-loop	TCGCACTGGATACGACTTGGCA
sst-miRNA-R	AGTGCAGGGTCCGAGGTATT
sst-GAPDH-F	ACTGGTGCAGCTAAAGCTGTAGG
sst-GAPDH-loop	GTCGTATCCAGTGCAGGGTCCGAGG

样品名称 Sample name	原始序列 Raw sequence	总读数 Total bases	GC含量/% GC content/%	质量值大于20的碱基数占比% Q20/%	质量值大于20的碱基数占比% Q20/%	总匹配数 Total mapped	总匹配率/% Total mapped/%
iL₃-1	24 795 684	0.553 G	50.19	99.56	95.79	22 785 621	92.0
iL₃-2	24 937 291	0.550 G	48.53	99.49	95.04	22 400 131	89.8
iL₃-3	23 295 886	0.526 G	47.99	99.14	96.47	19 707 809	84.9
pF-1	23 853 139	0.533 G	49.26	99.52	95.90	13 335 379	56.2
pF-2	23 698 225	0.529 8 G	50.98	99.48	94.92	12 059 955	51.1
pF-3	23 735 746	0.544 G	49.43	99.43	94.78	12 608 177	53.7

miRNA名称miRNA name	log₂（差异倍数） log₂ (Fold change)	iL₃ TPM	pF TPM
sst-novel-108	-2.408	132 509	24 969
sst-novel-16	2.022	3 213	13 050
sst-novel-134	-1.600	21 202	6 996
sst-novel-117	-2.052	12 863	3 102
sst-novel-22	-1.799	3 548	1 020
sst-novel-18	1.983	2 330	9 209
sst-novel-9	-1.661	13 859	4 383
sst-novel-106	1.453	14 141	38 714
sst-novel-7	-2.531	18 005	3 115
sst-novel-120	1.326	2 698	6 766
sst-novel-19	-1.108	4 890	2 268
sst-novel-10	-1.202	13 289	5 774