| Alveolar echinococcosis(AE)is a serious zoonotic disease caused by the larva of Echinococcus multilocularis mostly residing in the liver and lung in humans and animals,threatening human health.Because of the long latent period and invasive growth of larvae,the current fatality rate of AE is still very high and should not be underestimated.Micro RNAs,a class of regulatory molecules,which mainly bind to the 3’UTR of target m RNA.It is shown that miR-71 is only expressed in deuterostomes,suggesting that emu-miR-71 can be used as a potential drug target for anti-alveolar echinococcosis.The effects of emu-miR-71 on the peritoneal macrophages E.multilocularis-infected mice were explored.Firstly,q PCR showed that emu-miR-71 were existed in infected mice’s abdominal cavity.But its origin and biological function are unknown.So the exosomes from the peritoneal cavity of infected mice were isolated,and the parasite-derived exosomes were identified using the gold-labeled 14-3-3antibody.The PKH26-labeled exosomes were co-incubated with separated peritoneal macrophages.The results showed that exosomes could be obviously internalized by peritoneal macrophages of flow cytometry and laser confocal microscopy,which suggests that emu-miR-71 exerts its biological functions after it is transported and released into the host body through exosomes.Finally,emu miR-71 mimics and NC were transfected into isolated mouse peritoneal macrophages and q PCR found that the expression levels of IL-1α,IL-1β,IL-4 and other genes,such as RIPK1,NF-κB and TICAM2 in LPS/TLR4 pathway,were significantly down-regulated after emu-miR-71 mimics transfected(p<0.05).These results suggest that emu-miR-71 plays the biological functions by affecting the expression of the above genes.Previous studies have found that there is a certain abundance of emu-miR-71 in the infected liver through RNA-seq and other methods,but its function is unknown.For further verification,we isolated hepatocytes,hepatic stellate cells and Kupffer cells,and the expression of emu-miR-71 in each type of cells was detected via q PCR.The results showed that emu-miR-71 was highly expressed in hepatic stellate cells and hepatocytes.Then PKH26-labeled exosomes and the hepatocytes were co-incubated,and flow cytometry showed that hepatocytes could internalize exosomes.To analyze the effect of over-expressed emu-miR-71 on the hepatocyte’s proteome,emu-miR-71 mimics and NC were transfected into NCTC 1649.The results showed that the specific proteins of the emu-miR-71 mimics and NC groups were 636 and 302,respectively,while 706 were shared in common.KEGG showed that more than 150 proteins were involved in metabolic pathways,followed by ribosomal and carbon metabolism pathways.Using q PCR,only aldolase(Aldoa)and nucleolin(Ncl)genes were verified to be of ectopic expression(p<0.01),but their biological functions need further studies.For the down-regulation of emu-miR-71 in infected liver,we synthesized emu-miR-71 inhibitor packaging vector(AAV8-miR-71-sponge)and injected via mouse tail vein.Three months after E.multilocularis infection,the mice were sacrificed and the weight and cysts’ number in the liver and abdominal cavity were all detected.The results showed that the AAV-miR-71-sponge group had a significant difference in the weight of abdominal cysts compared to NC group(p<0.05),while there were no significant differences in the others,indicating that emu-miR-71 inhibitors can inhibit the growth of E.multilocularis residing the abdominal cavity.In summary,emu-miR-71 is released into peritoneal macrophages through exosomes during E.multilocularis infection,which may affect the immunological functions by regulating cytokines and LPS/TLR4 signaling pathway.Moreover,emu-miR-71 inhibitor can inhibit the growth of E.multilocularis residing the abdominal cavity and has potential application values. |
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