收稿日期: 2022-10-26
修回日期: 2022-12-02
网络出版日期: 2023-09-06
基金资助
中央高校基本科研业务费专项基金(2662017PY084)
Identification of miRNAs in the infectious third stage larvae and parasitic female adult of Strongyloides stercoralis
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)
目的 鉴定粪类圆线虫感染性Ⅲ期幼虫（iL3）和寄生性雌虫（pF）的微小RNA（miRNA），分析差异表达miRNA及其靶基因的功能。 方法 从粪类圆线虫感染犬粪便中收集iL3。取4 000~5 000条iL3于颈后皮下注射感染健康犬，感染后21 d，从肠道中采集pF。提取iL3和pF的总RNA并测序。测序数据与粪类圆线虫基因组和鼠类圆线虫miRNA比对筛选保守的miRNA，用miRDeep2通过颈环结构分析、成熟序列比对筛选miRNA。分析iL3和pF的miRNA表达谱，筛选差异表达miRNA，预测差异表达miRNA的靶基因，对log2（差异倍数）> 1和每百万转录本上的读数（TPM）> 1 000的未注释miRNA的靶基因功能进行分析。用ClusterProfiler对差异表达miRNA靶基因进行GO富集分析。选取11个未注释或保守的差异表达miRNA进行实时荧光定量PCR（qRT-PCR）验证，以粪类圆线虫甘油醛-3-磷酸脱氢酶基因（GenBank登录号：BI773092.1）为参照基因，计算差异表达miRNA的相对转录水平。 结果 共鉴定出265个miRNA，包括130个保守的miRNA和135个未注释的miRNA。其中，134个miRNA在iL3和pF之间差异表达（包含77个保守的miRNA和57个未注释的miRNA），251个为iL3和pF所共有，10个为iL3特有，4个为pF特有。差异表达miRNA靶基因预测结果显示，57个未注释的miRNA中，有12个差异倍数 > 2且TPM > 1 000，有248个靶基因与秀丽隐杆线虫同源，其中35.08%（87/248）的靶基因与发育相关，其余与应激、运动和蜕皮等相关。大部分在pF高表达未注释的miRNA靶向SSTP_0000114700。差异表达miRNA靶基因的GO分析结果显示，共5 595个靶基因被富集，其中富集靶基因数居前5位的分别为膜组成成分（1 821个）、核酸结合（561个）、细胞核（450个）、蛋白质水解作用（365个）和信号传导（284个）。qRT-PCR验证结果显示，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的log2（差异倍数）分别为-2.13、6.39、4.46、-3.69、-3.69、2.34、-2.48、-2.41、-2.30、2.25、-3.32，转录组分析获得的log2（差异倍数）分别为-3.05、4.98、4.07、-4.9、-3.66、0.98、-3.79、-2.61、-0.99、0.63、-1.55。qRT-PCR与转录组分析获得的上调、下调趋势结果一致。 结论 获得粪类圆线虫感染性Ⅲ期幼虫和寄生性雌虫的miRNA表达谱和差异表达miRNA，差异表达miRNA与粪类圆线虫的生长发育和繁殖功能相关。
覃裴溪 , 周彩显 , 鲁志刚 , 张碧瀛 , 周涛勋 , 胡敏 . 粪类圆线虫感染性Ⅲ期幼虫和寄生性雌虫miRNA的鉴定[J]. 中国寄生虫学与寄生虫病杂志, 2023 , 41(4) : 412 -420 . DOI: 10.12140/j.issn.1000-7423.2023.04.003
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.
| [1] | Beknazarova M, Whiley H, Ross K. Strongyloidiasis: a disease of socioeconomic disadvantage[J]. Int J Environ Res Public Health, 2016, 13(5): 517. |
| [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. |
| [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. |
| [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. |
| [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. |
| [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. |
| [8] | Hammond SM. An overview of microRNAs[J]. Adv Drug Deliv Rev, 2015, 87: 3-14. |
| [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. |
| [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. |
| [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 |
| [12] | Ulusan Ba?c? ?, Caner A. The role of microRNAs in parasitology[J]. Turkiye Parazitol Derg, 2020, 44(2): 102-108. |
| [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. |
| [14] | Ma GX, Luo YF, Zhu HH, et al. microRNAs of Toxocara canis and their predicted functional roles[J]. Parasit Vectors, 2016, 9: 229. |
| [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. |
| [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. |
| [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. |
| [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. |
| [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. |
| [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. |
| [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. |
| [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. |
| [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. |
| [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. |
| [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. |
| [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. |
| [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. |
| [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. |
| [31] | Davis MW, Birnie AJ, Chan AC, et al. A conserved metalloprotease mediates ecdysis in Caenorhabditis elegans[J]. Development, 2004, 131(23): 6001-6008. |
| [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. |
| [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. |
| [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. |
| [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. |
| [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. |
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