CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES >

2023 , Vol. 41 >Issue 4: 412 - 420

DOI: https://doi.org/10.12140/j.issn.1000-7423.2023.04.003

ORIGINAL ARTICLES

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

  • QIN Peixi ,
  • ZHOU Caixian ,
  • LU Zhigang ,
  • ZHANG Biying ,
  • ZHOU Taoxun ,
  • HU Min
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  • 1 National Key Laboratory of Agricultural Microbiology Core Facility, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, Hubei, China
    2 Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridgeshire, CB10 1SA, UK

Received date: 2022-10-26

  Revised date: 2022-12-02

  Online published: 2023-09-06

Supported by

Fundamental Research Funds for the Central Universities(2662017PY084)

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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

Cite this article

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 . DOI: 10.12140/j.issn.1000-7423.2023.04.003

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