Regulation Of MicroRNAs In Schistosoma Japonicum Infection With Yellow Cattle And Water Buffalo

Yellow cattle(Bos taurus)and water buffalo(Bubalus bubalis)are important natural reservoir hosts and the main transmission sources of Schistosoma japonicum in endemic areas of China.The susceptibility of the two hosts to schistosome infection is different,which leads to great difference in the worm growth,development and survival,as well as pathological damage to the hosts themselves.To investigate micro RNAs(miRNAs)involved in the regulation of schistosome development and the interplay between schistosomes and their natural final hosts,we compared miRNA expression profiles of the adult worms,plasma and peripheral blood mononuclear cells(PBMC)of yellow cattle(susceptible host)and water buffalo(non-susceptible host)at three sampled time by using high-throughput sequencing with Illumina Hiseq Xten.Three sampled time of plasma and PBMC were marked as T1(before infection),T2(two weeks post infection)and T3(six weeks post infection).The biological functions of eukaryotic initiation factor 4(eIF4A)of S.japonicum were also studied.These studies may provide new ideas for screening vaccine candidate molecules and new drug targets against schistosomiasis.1.Comparative analysis of micro RNA expression profiles of adult S.japonicum isolated from water buffalo and yellow cattleThe miRNA expression profiles of adult schistosomes derived from yellow cattle and water buffalo were compared by using high-throughput sequencing.S.japonicum from water buffalo and yellow cattle yielded 63.78 million and 63.21 million reads,respectively.A total of 206 miRNAs were identified by bioinformatics analysis,including 79 previously annotated miRNAs of S.japonicum,127 miRNAs that matched with the S.japonicum genome and were highly similar to the annotated miRNAs from other organisms.Among of them,five known miRNAs(sja-miR-124-3p,sja-miR-219-5p,sja-miR-2e-3p,sja-miR-7-3pand sja-miR-3490)were significantly up-regulated in worms derived from water buffalo.Ten novel miRNAs were also significantly differentially expressed in the schistosomes from these two hosts.The expression trend of sja-miR-124-3p in worms recovered from water buffalo and yellow cattle was consistent with that in worms derived from different susceptible rodent hosts.It was found that sja-miR-124-3p was also involved in the regulation of eIF4 A of schistosome.Dual-luciferase reporter assay confirmed that sja-micro RNA-219-5p could bind to specific sequence of target gene Hsc20.A total of 268 potential target genes were predicted for these five differentially expressed miRNAs.GO analysis revealed these targets were primarily involved in pharyngeal muscle development,meiotic cytokinesis,establishment of meiotic spindle localization,cytoskeleton-dependent intracellular transport,r RNA processing,protein transport,reproduction and muscle cell fate specification.sja-miR-124-3p and sja-miR-219-5p may affect the survival and development of worms by regulating their nervous system development and stress response in these two natural final hosts.2.Comparative analysis of plasma miRNA expression profiles of water buffalo and yellow cattle during S.japonicum infectionThe miRNA expression profiles of plasma from water buffalo and yellow cattle at three sampled time during S.japonicum infection were investigated by using high-throughput sequencing.Plasma samples from water buffalo and yellow cattle yielded352,727,942 reads in total and each sample possess an average of 14,696,997 reads.The obtained clean reads were mapped to the yellow cattle and water buffalo genome with a mapping ratio of 90.80% and 99.60%,respectively.All the 1,030 miRNAs which have been annotated in the miRbase before have been detected in all the plasma samples derived from yellow cattle and water buffalo.In addition,444 and 438 new miRNAs in yellow cattle and water buffalo plasma,respectively were discovered through bioinformatic analysis.Compared with T1,3 and 9 miRNAs were found to be unique differentially expressed in yellow cattle and water buffalo plasma,respectively at T2.9 miRNAs were present as common differentially expressed ones between yellow cattle and water buffalo plasma at T3,while 39 and 23 miRNAs were unique differentially expressed in yellow cattle and water buffalo plasma,respectively at T3.Compared with T2,4 miRNAs werepresent as common differentially expressed,while 35 miRNAs and 5 miRNAs were identified as unique differentially expressed in yellow cattle and water buffalo plasma,respectively at T3.GO and KEGG analysis of the targets of the differentially expressed miRNAs indicated that many important biological pathways are triggered.During the early stage of schistosome infection,the miRNAs involved in fatty acid metabolism and insulin signaling pathway were significantly differentally expressed in yellow cattle and water buffalo plasma,which may affect the survival and development of worms in two hosts.3.Comparative analysis of PBMC miRNA expression profiles of water buffalo and yellow cattle during S.japonicum infection.High-throughput sequencing with Illumina Hiseq Xten was applied to investigate the miRNAs expression in PBMC derived from yellow cattle and water buffalo at three sampled time.All PBMC samples from water buffalo and yellow cattle yielded228,483,170 reads in total and each sample possess an average of 9,520,132 reads.All the1,030 known miRNAs in the miRbase have been detected in all the PBMC samples derived from two groups.In addition,234 and 228 new miRNAs were discovered in yellow cattle and water buffalo PBMC,respectively.Compared with T1,45 miRNAs were present as common differentially expressed,while 111 and 69 miRNAs were unique differentially expressed in yellow cattle and water buffal PBMC,respectively at T3.Compared with T2,64 miRNAs were identified as common differentially expressed,while 147 and 36 miRNAs were identified as unique differentially expressed in yellow cattle and water buffalo PBMC,respectively at T3.GO and KEGG analysis of the differentially expressed miRNAs suggested that many important biological pathways are involved.In the acute infection stage of schistosome,the miRNAs involved in Th1/Th2 cytokine regulation and Toll-like receptor signaling pathway were significantly differentally expressed in yellow cattle and water buffalo PBMC,which may affect the host’s immune response.4.Cloning,expression and characterization of the eukaryotic translation initiation factor eIF4 A of S.japonicum.The DNA encoding eIF4 A was amplified by PCR according to Gen Bank accession:FN315265.1.The recombinant plasmid p ET-28a(+)-eIF4 A was transformed into E.coli BL21(DE3).The target protein was expressed by using conventional Isopropylβ-D-Thiogalactoside(IPTG)induction,and purified by His-tag magnetic beads.The recombinant protein and Sj-eIF4 A in the worm protein were further analyzed and comfirmed by Western blot.The transcription levels of Sj-eIF4 A in worms derived from different developmental stages and different suitable hosts were detected by q RT-PCR.The RNA interference were performed on 21-day schistosomula in vitro and in vivo,and the effects of RNA interference on the reproduction and structure of the worms were observed by scanning electron microscopy(SEM).The results showed that the length of Sj-eIF4 A was 1179 bp,encoding a polypeptide of 392 amino acids.The fusion protein was approximately 46 k Da.Real-time PCR analysis revealed that Sj-eIF4 A was expressed at different developmental stages of the tested schistosomes.The transcriptional level of Sj-eIF4 A was peak in the miracidium stage,followed by 14 d and 42 d female worms.The transcription level of Sj-eIF4 A in worms derived from susceptible hosts yellow cattle and BALB/c were higher than those in non-susceptible hosts water buffalo and Wistar rats.Compared with the diethyl pyrocarbonate(DEPC)group,BALB/c treated with SjeIF4 A specific small interfering RNA(si RNA)in vivo showed a 20.63% worms burden reduction(P<0.05)and 31.21% hepatic egg burden reduction(P<0.01),respectively.Compared with the blank group,the worms treated with Sj-eIF4 A si RNA in vitro lead to abnormal egg-laying.SEM revealed that SjeIF4 A silience in schistosomes caused significant alterations in the ventral sucker and the tegumental of female and male worms.In summary,the miRNA expression profiles in schistosomes derived from yellow cattle and water buffalo,and in PBMC and plasma from these two natural final hosts pre/post infection with S.japonicum were compared and analyzed.The cloning,expression and gene characteristics analysis of eIF4 A of S.japonicum were also performed.Many important parasite-derived and host-derived miRNAs associated with worm development and host immune response had been found.The biological functions of Sj-eIF4 A were preliminary explored.This study provided valuable information for revealing the interaction between different suitablility host and schistosome,as well as provided new ideas for screening vaccine candidate molecules or new drug targets against schistosome.