CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES >

2020 , Vol. 38 >Issue 2: 188 - 193

DOI: https://doi.org/10.12140/j.issn.1000-7423.2020.02.009

ORIGNAL ARTICLES

Analysis of miR-71 secretion and expression characteristics of Echinococcus granulosus protoscolex

  • Lu-jun YAN ,
  • Ya-ting LI ,
  • Jun-jun DING ,
  • Jing YANG ,
  • Ya-dong ZHENG ,
  • Yi-xia CHEN
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  • 1 Life Science and Engineering College of Northwest University for Nationalities, Lanzhou 730030, China
    2 State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China
    3 Huchuan Animal Husbandry Center, Zhangjiachuan Hui Autonomous County, Tianshui 741000, China

Received date: 2019-11-19

  Online published: 2020-05-11

Supported by

Supported by the National Natural Science Foundation of China(U1703104);the Cultivation Project of Innovation Team for Basic Scientific Research Business Expenses of Central Universities in Northwest Minzu University(31920190027);the National Key Basic Research and Development Plan 973 Plan Project(No. 2015CB150300)

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Abstract

Objective To analyze the secretion and expression characteristics of miR-71 of Echinococcus granulosus protoscolex under stimulation by insulin, albendazole and artificial gastric fluid.Methods E. granulosus cysts in sheep liver and lung tissues were collected at a slaughterhouse in Xinjiang, China. The E. granulosus cysts were fixed, embedded and sectioned, followed by hybridization with a miR-71 probe. The sections were then incubated with antibody labeled with fluorescein isothiocyanate (FITC), and then stained with a nucleic acid dye 4’, 6-diamidino-2-phenylindole (DAPI) to visualize the allocation of miR-71 in E. granulosus protoscoleces and cysts wall with fluorescence microscopy, analyzing the distribution of miR-71 in the protoscoleces at various developmental stages, for ascertaining the expression of miR-71 in the protoscoleces. In addition, the protoscoleces were cultured in the presence of insulin, albendazole and artificial gastric fluid and the supernatant was collected to isolate exosomes by differential centrifugation. The morphology of exosomes was observed under a transmission electron microscope. The particle-size distribution of the exosomes was measured by a nanoparticle size analyzer. The enolase and 14-3-3 protein were used as biomarkers for identification of exosome by Western blotting. The abundance of miR-71 in exosomes was determined by real-time quantitative PCR (qPCR).Results In situ hybridization showed that miR-71 was expressed in the middle and late developmental stages of protoscoleces and in the germinal layer of cyst wall of E. granulosus. The exosomes had a size of 48.9 nm, 49.7 nm and 65.4 nm in the groups of artificial gastric fluid, insulin, and albendazole, respectively, all within the range of 40-120 nm, and morphologically shaped as a membrane-enveloped sphere. Western blotting detected the presence of enolase and 14-3-3 protein in exosomes, demonstrating successful extraction of exosomes. qPCR showed that the expression level of miR-71 in exosomes secreted from protoscoleces in the test group with artificial gastric fluid, insulin, and albendazole was 1.84, 1.87 and 2.38 folds of the control group (t = 12.8, 26.7 and 29.3, P < 0.01), respectively.Conclusion miR-71 is widely expressed in protoscoleces and cyst wall of E. granulosus, and could be secreted via exosomes. The expression and secretion of miR-71 can be upregulated by stimulations with insulin, artificial gastric fluid and albendazole.

Key words： Echinococcus granulosus; Protoscolex; miR-71; Expression characteristics; Exosome

Cite this article

Lu-jun YAN , Ya-ting LI , Jun-jun DING , Jing YANG , Ya-dong ZHENG , Yi-xia CHEN . Analysis of miR-71 secretion and expression characteristics of Echinococcus granulosus protoscolex[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2020 , 38(2) : 188 -193 . DOI: 10.12140/j.issn.1000-7423.2020.02.009

References

[1] Liu P, Li JH, Li Y , et al. The epidemic situation and causative analysis of echinococcosis[J]. China J Animal Quar, 2016,33(1):48-51. (in Chinese)
[1] ( 刘平, 李金花, 李印 , 等. 包虫病病原在我国的流行现状及成因分析[J]. 中国动物检疫, 2016,33(1):48-51.)
[2] Wu WP, Wang H, Wang Q , et al. A nationwide sampling survey on echinococcosis in China during 2012-2016[J]. Chin J Parasitol Parasit Dis, 2018,36(1):1-14. (in Chinese)
[2] ( 伍卫平, 王虎, 王谦 , 等. 2012-2016年中国棘球蚴病抽样调查分析[J]. 中国寄生虫学与寄生虫病杂志, 2018,36(1):1-14.)
[3] Qian MB, Chen J, Bergquist R , et al. Neglected tropical diseases in the People’s Republic of China: progress towards elimination[J]. Infect Dis Poverty, 2019,8(1):86.
[4] Wei YH, Hu Y, Cao JP . Progress toward development of a vaccine against Echinococcus granulosus infection[J]. Chin J Parasitol Parasit Dis, 2019,37:97-101. (in Chinese)
[4] ( 魏玉环, 胡媛, 曹建平 . 抗细粒棘球绦虫疫苗的研究进展[J]. 中国寄生虫学与寄生虫病杂志, 2019,37(1):97-101.)
[5] Eckert J, Deplazes P . Biological, epidemiological, and clinical aspects of echinococcosis, a zoonosis of increasing concern[J]. Clin Microbiol Rev, 2004,17(1):107-135.
[6] Stefani G, Slack FJ . Small non-coding RNAs in animal development[J]. Nat Rev Mol Cell Biol, 2008,9(3):219-230.
[7] Zhu SL, Wang S, Lin Y , et al. Release of extracellular vesicles containing small RNAs from the eggs of Schistosoma japonicum[J]. Parasit Vectors, 2016,9(1):574.
[8] Liu JT, Zhu LH, Wang JB , et al. Schistosoma japonicum extracellular vesicle miRNA cargo regulates host macrophage functions facilitating parasitism[J]. PLoS Pathog, 2019,15(6):e1007817.
[9] Buck AH, Coakley G, Simbari F , et al. Exosomes secreted by nematode parasites transfer small RNAs to mammalian cells and modulate innate immunity[J]. Nat Commun, 2015,6:8772.
[10] Tritten L, Burkman E, Moorhead A , et al. Detection of circulating parasite-derived microRNAs in filarial infections[J]. PLoS Negl Trop Dis, 2014,8(7):e2971.
[11] Hsieh YW, Chang C, Chuang CF . The microRNA mir-71 inhibits calcium signaling by targeting the TIR-1/Sarm1 adaptor protein to control stochastic L/R neuronal asymmetry in C. elegans[J]. PLoS Genet, 2012,8(8):e1002864.
[12] 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.
[13] Zheng YD, Cai XP, Bradley JE . MicroRNAs in parasites and parasite infection[J]. RNA Biol, 2013,10(3):371-379.
[14] Quintana JF, Makepeace BL, Babayan SA , et al. Extracellular Onchocerca-derived small RNAs in host nodules and blood[J]. Parasit Vectors, 2015,8:58.
[15] Hoy AM, Lundie RJ, Ivens A , et al. Parasite-derived microRNAs in host serum as novel biomarkers of helminth infection[J]. PLoS Negl Trop Dis, 2014,8(2):e2701.
[16] Valadi H, Ekstr?m K, Bossios A , et al. Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells[J]. Nat Cell Biol, 2007,9(6):654-659.
[17] Zheng YD, Guo XL, Su M , et al. Regulatory effects of Echinococcus multilocularis extracellular vesicles on RAW264.7 macrophages[J]. Vet Parasitol, 2017,235:29-36.
[18] Ding J, He G, Wu J , et al. miRNA-seq of Echinococcus multilocularis extracellular vesicles and immunomodulatory effects of miR-4989[J]. Front Microbiol, 2019,10:2707.
[19] Su M, Guo X L, Yang J , et al. Screening and applications of qPCR primers for apomucin gene of Echinococcus multilocularis[J]. Chin J Parasitol Parasit Dis, 2016,34(5):394-398. (in Chinese)
[19] ( 苏梦, 郭小腊, 杨静 , 等. 多房棘球绦虫apomucin基因qPCR引物的筛选及潜在应用[J]. 中国寄生虫学与寄生虫病杂志, 2016,34(5):394-398.)
[20] Wen LM, Lü GD, Zhao J , et al. Molecular cloning and characterization of ribosomal protein RPS9 in Echinococcus granulosus[J]. J Parasitol, 2017,103(6):699-707.
[21] Smith-Vikos T, de Lencastre A, Inukai S , et al . MicroRNAs mediate dietary-restriction-induced longevity through PHA-4/FOXA and SKN-1/Nrf transcription factors[J]. Curr Biol, 2014,24(19):2238-2246.
[22] Guo XL, Zhang XY, Yang J , et al. Suppression of Nemo-like kinase by miR-71 in Echinococcus multilocularis[J]. Exp Parasitol, 2017,183:1-5.
[23] Kanwar JR, Vinayak VK . Isolation & immunochemical characterization of diagnostically relevant antigens of Echinococcus granulosus[J]. Indian J Med Res, 1993,97:75-82.
[24] Ceballos L, Virkel G , Elissondo, et al. A pharmacology-based comparison of the activity of albendazole and flubendazole against Echinococcus granulosus metacestode in sheep[J]. Acta Trop, 127(3):216-225.
[25] Pan W, Shen Y, Han X , et al. Transcriptome profiles of the protoscolexes of Echinococcus granulosus reveal that excretory-secretory products are essential to metabolic adaptation[J]. PLoS Negl Trop Dis, 2014,8(12):e3392.
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