Preliminary Analysis Of Secreted Expression Characteristics And Biological Activity Of Echinococcus Granulosus MiR-71

Cystic hydatid disease is a zoonotic parasitic disease caused by the mid-stage larvae of E.granulosus.It is one of the diseases that poses a serious threat to global public health.It is caused about three million Yuan loss every year in China alone.E.granulosus has a global distribution.China is mainly distributed in agricultural,pastoral and semi-agricultural areas in Qinghai,Gansu,Tibet,Xinjiang,and Inner Mongolia.Many studies in recent years shows that microRNAs(miRNAs)molecules encoded by parasites can promote host parasites by regulating the host's immune function.Therefore,research on the secretory expression characteristics and biological functions of miRNA molecules in E.granulosus is great to find new targets for anti-parasitic drugs and diagnostic reagents.In view of this,this research has mainly carried out the following aspects:1.In order to analys the tissue distribution and secretion expression of miR-71 in E.granulosus,a miR-71 probe labeled with Digoxin was synthesized,and miR-71 Expression characteristics in E.granulosus was analyzed by in-situ hybridization.;The protoscolexes were vitro culture by medium adding with insulin,pepsin,and albendazole to simulate the environment of host liver,intestinal tract and administration conditions,and then exosomes were collected andidentification.;miR-71 expression abundance in exosome was detected by qPCR.The results of in-situ hybridization analysis showed that miR-71 was widely distributed in the vesicle wall and protoscolexes of E.granulosus,and was expressed in the protoscolexes in the middle and late stages of development.Transmission electron microscope observation and nanoparticle size analyzer detection of exosomes showed that the exosomes secreted by the protoscolexes of the pepsin group,the insulin group,and the albendazole group were in the shape of a membrane-encapsulated vesicle,the particle sizes were 48.9,49.7 and 65.4nm.By Western blotting,theexosome biomarker of enolase and 14-3-3 protein were detected.The qPCR results showed that the expression of miR-71 in the exosomes of the pepsin group,insulin group,and albendazole group were all 1.84,1.87,and 2.38 times higher than those in the control group(P < 0.01).2.To explore the internalization of exosomes,The PKH-26 dye were used to stain the exosomes and co-incubated with mouse macrophages RAW264.7,mouse liver cells NCTC1469 and human kidney epithelial cells HEK-293 t,and then the location of exosomes was observed using a laser confocal microscope,and the internalization efficiency of exosomes was analyzed by flow cytometry.The results showed that exosomes were internalized into the cytoplasm by RAW264.7,NCTC1469 and HEK-293 t cells,and a small amount of exosomes also existed on the cell wall of RAW264.7 and HEK-293 t cells.Compared with the control group,exosomes can be effectively internalized into RAW264.7,NCTC1469 and HEK-293 t cells with internalization rates of 72.1 %,18.2 % and 18.5 %,respectively.Moreover,the internalization rate of exosomes in RAW264.7 cells was significantly higher than that in NCTC1469 and HEK-293 t cell groups,with significant differences(P <0.01).3.In order to preliminary analyze the biological activity of miR-71 in exosomes,the target gene NLK 3?UTR of miR-71 was used to construct wild-type and mutant fluorescent reporter plasmid pCMV-eGFP-NLK.The miRNA negative control,MiR-71 mimics and exosomes were used co-transfected into HEK-293 t cells,and the average fluorescence intensity(MFI)of HEK-293 t cells was detected by flow cytometry.The results showed that compared with the control group,the MFI values of miR-71 mimics + exosomes co-transfection group in the pCMV-eGFP-NLK-WT experimental group were significantly reduced,with significant differences(P <0.01).There was no significant change in MFI value in the mutant pCMV-eGFP-NLK-Mut experimental group(P> 0.1).Conclusion: miR-71 is widely expressed in the protoscolexes and cyst wall of E.granulosus.Different environments in the host can stimulate expression of miR-71 in the protoscolexes and enter the host cytoplasm through exosomes,especially in immune cells.miR-71 in exosomes is capable of binding to its known target genes.