Identification Of Target Genes Of Mir-26a-5p Associated With Echinococcus Multilocularis Infection And Its Effect On Apoptosis Of Spleen Lymphocytes

Alveolar echinococcosis in humans and animals is caused by the metacestode stage of tapeworm parasite Echinococcus multilocularis,which seriously threaten to human health and development of animal husbandry.Micro RNAs(miRNAs)are a class of non-coding small RNAs with the function of post-transcriptional gene expression regulation.miRNAs play key roles in the regulation of cell development and activating immune response.Studies have shown that miRNAs can promote or inhibit the parasitism in the host by regulating host immune response.Therefore,screening and functional studies of miRNAs associated with E.multilocularis infection will be of great significance for finding novel anti-parasite drugs and diagnostic targets.In view of this,this paper has carried out research work in the following aspects:1.Screening and dynamic analysis of miRNAs associated with E.multilocularis infection.High-throughput sequencing technology was used to screen for differentially expressed miRNAs in spleen lymphocytes of mice before and after infection with E.multilocularis.The dynamic expression of the most significantly differentially expressed miRNAs during infection was analyzed by q PCR.There were 195 differentially expressed miRNAs during E.multilocularis infection.Among them,the differential expression of miR-26a-5p was the most significantly differentially expressed,and its expression level was significantly down-regulated at 30,60 and 90 days after infection E.multilocularis,respectively(P < 0.05).2.Identification of target genes of miR-26a-5p.First,the potential target genes of miR-26a-5p were predicted by bioinformatics methods.Then,the transcription and protein expression of potential target genes were detected in splenic lymphocytes at different days post-infection with E.multilocularis by q PCR and western blot.Finally,the target gene of miR-26a-5p was verified by double luciferase reporting system.The result showed that miR-26a-5p had nine potential target genes,including EZH2,WNK1,PATZ1,ZFP608,DDX17,CTSL,FNIP1,EP300,and FA2 H.The result showed that the transcription and protein expression of EZH2 were significantly up-regulated at 30,60 and 90 days after infection(P < 0.01).miR-26a-5p could combine with the 3’UTR of EZH2 m RNA.3.The effect of miR-26a-5p on splenic lymphocyte apoptosis during E.multilocularis infection.First,flow cytometry was used to detect the apoptosis of splenic lymphocytes in mice at different stages of E.multilocularis and the splenic lymphocytes of overexpression of miR-26a-5p.Finally,in order to preliminarily explore the mechanism of miR-26a-5p on apoptosis,spleen lymphocytes of healthy mice were incubated with E.multilocularis exosomes in vitro for 24 h.At the same time,the E.multilocularis exosomes were injected into normal mice through the caudal vein,and spleen lymphocytes were isolated after 24 hours of the injection.The expressions of miR-26a-5p and EZH2 were detected by q PCR.Apoptosis was detected by flow cytometry.The results showed that the apoptosis levels of splenic lymphocytes were significantly decreased at 30,60 and 90 days after E.multilocularis infection(P < 0.05).The apoptosis level was significantly up-regulated in spleen lymphocytes of healthy mice transfected with miR-26a-5p(P < 0.05).E.multilocularis exosomes could inhibit the miR-26a-5p expression andup-regulate the EZH2 expression.Exosomes treatments significantly inhibited the apoptosis levels of splenic lymphocytes in vivo and in vitro(P < 0.01).Conclusion: The miR-26a-5p expression in splenic lymphocytes may be inhibited though E.multilocularis–derived exosomes during the infection,resulting in the upregulation of its target gene EZH2,thus inhibiting the apoptosis of splenic lymphocytes.