Immunomodulatory Effects Of Murine MiR-146a-5p In Echinococcus Multilocularis Infection

Echinococcosis is an important zoonotic parasitic disease.It is mainly caused by the larvae of Echinococcus spp,which primarily resides in the liver and lungs of cattle,sheep and humans.The larvae of Echinococcus multilocularis is also parasitic in rodents and humans,causing alveolar echinococcosis(AE).MicroRNAs(miRNAs)are a class of small regulatory non-coding RNAs.A large amount of data indicates that they play an important role in the growth,development,infection and immunity of parasites.Therefore,the research on the mouse miRNAs in the infectious process of E.multilocularis can not only reveal its pathogenic mechanisms,but also has important significance for finding new drug targets.For this,we concentrated on the following aspects in the study:The BALB/c mouse infection model of E.multilocularis was established.The indirect ELISA and dissection results showed that the infection rate of mice remained at about 38%.Based on this,flow cytometry was used to first detect the apoptosis levels of the spleen cells of mice 3 months post infection by E.multilocularis.The results showed that the level of apoptosis of mice spleen cells was significantly decreased(P < 0.05)in the experimental group(intraperitoneal injection of E.multilocularis protoscoleces)compared with the control group(intraperitoneal injection of PBS).In order to explore the possible molecular mechanism of spleen cell apoptosis changes,using high-throughput RNA-seq sequencing technology to analyze the expression profile of small RNA in the spleen cells,it was found that seven miRNAs were differentially expressed,such as miR-124-3p,miR-9-5p,miR-129-2-3p,miR-1a-3p ? miR-128-3p(up-regulated),miR-16-5p and miR-146a-5p(down-regulated).Further bioinformatics analysis revealed that 13 target genes of miR-146a-5p were related to immune-related or apoptosis,such as TLR3,IRAK1,NFAM1,IRAK2,TRAF6 and BAG-1 and so on.Therefore,the miR-146a-5p molecule was selected to conduct in-depth studies.The mouse spleen cells were isolated in different infection periods and qRCR was used to determine the level of miR-146a-5p expression.The results showed that miR-146a-5p was significantly down-regulated in the second and third month after infection.This expression trend is consistent with the RNA sequencing results.But the transcription level did not change at the first month after infection.All results indicated that miR-146a-5p showed dynamic changes during the infection of E.multilocularis.To further validate the possible immunomodulatory effects of miR-146a-5p,the murine macrophage cell line RAW 264.7 was transfected with miR-146a-5p negative control,mimics and inhibitors,respectively.The transcription level of cytokines,the key genes in TLR4/LPS signal pathway,iNOS gene and NO secretion were determined.These data illustrated that the mimics-transfection group and the inhibitors-transfected group showed opposite changes,which indicates that miR-146a-5p has immunomodulatory effects on murine macrophages.miR-146a-5p mimics and inhibitors were transfected into RAW 264.7 cells,and possible target BAG-1(anti-apoptotic gene)was detected by qPCR and Western blot.The BAG-1 in different infectious periods was also detected by qPCR.The results showed that BAG-1 was significantly up-regulated at second month and third month after infection,further suggesting that it may be the target gene of miR-146a-5p.The results of the dual luciferase gene reporter system revealed that miR-146a-5p specifically binds to the 3' UTR of the BAG-1 mRNA,thereby inhibiting its expression.The apoptosis level of mouse spleen cells was measured at first month and second month after infection.The results showed that the spleen apoptosis level was decreased compared with the negative control group at second month of infection.The isolated normal spleen cells were transfected with miR-146a-5p mimics and inhibitors,respectively.The apoptosis level of spleen cells in the mimics-transfected group was significantly increased compared with the inhibitors-transfected group.These results indicated that mmu-miR-146a-5p may regulate the immunity by negatively regulating the expression of the target gene Bag-1 and thus inhibiting apoptosis of spleen cells.In summary,the expression of miR-146a-5p in mouse spleen cells was dramatically down-regulated during the infection of E.multilocularis,resulting in a significant up-regulation of its target gene BAG-1 expression,and thus inhibiting spleen cell apoptosis and regulating immune responses.