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.

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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