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

doi:10.12140/j.issn.1000-7423.2020.02.009

Abstract

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

CLC Number:

R383.33

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.

Figures/Tables 3

References 25

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