| Taenia pisiformis cysticercosis is distributed globally,which cause significant economic losses to the rabbit breeding industry.Exosomes from T.pisiformis cysticercus have been reported to induce macrophages polarized to M2 phenotype,which is related to the immunosuppression caused by the metacestodes.Moreover,small RNA sequencing revealed that let-7-5p was abundant in exosomes from T.pisiformis cysticercus.So,do the exosomes from T.pisiformis cysticercus and let-7-5p participate in the immune regulation induced by worms? Can let-7-5p be used as a diagnostic target for T.pisiformis cysticercosis? This study has carried out the following work to solve the above two scientific problems:1.Tpi-let-7-5p from T.pisiformis cysticercus exosomes may be involved in T cell differentiationFirstly,using small RNA sequencing and proteomic analysis,we profiled the expression patterns of mi RNAs and proteins in rabbit peritoneal macrophages treated with T.pisiformis cysticercus-derived exosomes.A total of 21 significantly differentially expressed mi RNAs were found,including three upregulated worm-derived mi RNAs(tpi-let-7-5p,tpi-mi R-61-3p and tpi-mi R-71-5p).Function prediction showed that differentially expressed mi RNAs or proteins(a total of 599)were mainly involved in T cell differentiation,cell proliferation and apoptosis.Correlation network analysis revealed that NFk B2 may be the target gene of tpi-let-7-5p.2.Let-7-5p regulate the Treg cell differentiation through NFk B2The expressions of Th1,Th2,Th17 and Treg markers in PBLC of T.pisiformis-infected rabbits and Echinococcus multilocularis-infected mice were detected by q PCR.It was found that the infection of these two metacestodes can induce the differentiation of Treg cells in the host.Let-7-5p enriched in exosomes induced the expressions of Treg marker molecules TGF-β,IL-10 and Foxp3;while the exosomes collected after knockdown of worm-derived let-7-5p with RNA interference reversed the expressions of Treg marker molecules.Dual luciferase assay showed that NFk B2 was the target gene of let-7-5p,and Treg marker molecules were significantly up-regulated after knockdown of NFk B2,while Treg marker molecules were significantly down-regulated after overexpression of NFk B2.These results indicated that NFk B2 was involved in Treg cell differentiation.3.Knockdown of let-7-5p in mice infected with E.multilocularis can inhibit protoscolexs growthRecombinant adeno-associated virus type 8(AAV8-si-let-7-5p)expressing let-7-5p sponge sequence from metacestodes was injected into mice via tail vein to knockdown or inhibit the expression of let-7-5p.The results showed that,on the 15 th day after injection,AAV8-si-let-7-5p successfully targeted the mouse liver and caused knockdown of let-7-5p and up-regulation of NFk B2.At 3 months after infection,in the AAV8-si-let-7-5p group,the expression of let-7-5p in peripheral blood and spleen lymphocytes was decreased,while the expression of NFk B2 was increased,Treg marker molecules were significantly down-regulated,and cysts weight and protoscolexs number in liver were significantly lower than those of control group.It is suggested that knockdown of let-7-5p in mice infected with E.multilocularis can promote the expression of NFk B2,and thus inhibit the Treg cells differentiation and worm growth.4.RCA assisted CRISPR/Cas9 based on let-7-5p can be used as a universal diagnostic method for metacestodiasisIntergrating the advantages of CRISPR/Cas9 and RCA techniques,a diagnostic method based on let-7-5p was established,which can significantly distinguish let-7 from cestodes and other species,with the detection limit of 10 amol/L,the diagnostic sensitivity and specificity for rabbit cysticercosis and mouse echinococcosis multilocularis were 100%,97.67% and 100%,100%,respectively,and the plasma let-7-5p level was significantly positively correlated with worm load,suggesting that RCA assisted CRISPR/Cas9 based on let-7-5p can be used as a universal diagnostic method for early diagnosis(15 days)of metacestodiasis.In summary,the present study identified the differential expression profiles of mi RNAs and proteins in rabbit peritoneal macrophages treated with T.pisiformis cysticercus exosomes,and in vitro and in vivo studies suggested that let-7-5p targeted NFk B2 to regulate Treg cell differentiation,thereby inhibiting host immune response.RCA assisted CRISPR/Cas9 method established with let-7-5p as a target is a sensitive and specific diagnostic method,which is of great value for the early diagnosis and effective control of metacestodiasis. |
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