Identification Of MicroRNA Profile In Ascaris Lumbricoides And Ascaris Suum Adults And Stage-specific MicroRNAs In A. Suum And Preliminary Analysis The Functional Characterization Of Three MicroRNAs

The parasitic nematodes Ascaris lumbricoides and Ascaris suum are of great public health and economic significance, and the two taxa were proposed to represent a single species.We previously investigated the microRNA profile in male and female adults of A. suum. In this study, we compared the miRNA profiles of A. lumbricoides and A. suum female adults by Solexa deep sequencing combined with bioinformatics analysis and stem-loop real-time PCR, Using the A. suum genome as the reference genome, we obtained 171 and 494 miRNA candidates from A. lumbricoides and A. suum, respectively.Among which,74 miRNAs were shared between the two taxa,97 and 420 miRNAs were A. Lumbricoides and A.suum specific. Target and function prediction revealed a significant set of targets which are related to ovarian message protein, vitellogenin and chondroitin proteoglycan of the two nematodes. Enrichment analysis revealed that the percentages of most predicted functions of the miRNA targets were similar, with some taxon specific or taxon enhanced functions, such as different target numbers, specific functions (NADH dehydrogenase and electroncarrier functions), etc.This study characterized comparatively the miRNAs of adultA. lumbricoidesandA. suum,and the findings provide additional evidence that A. lumbricoidesandA. suumrepresent a single species,we also systematically investigated mircoRNA (miRNA) profile in this parasite in more detailed developmental stages (egg stage, the infected third larvae stage, L3 stage in lung, L4 stage, and L5 stage, as well as miRNA profiles data from female and male adult) using high-throughput RNA sequencing technology. we profiled and compared 7 miRNA profiles of the life-stage of A. suum, we identified various sets of stage and gender-specific miRNAs and targets, including conserved, embryo genesis related, development related, sex differentiation related, stage transition related miRNAs. From a total of 8 516 852 unique reads, we obtained 565,215, 157,128,174,486 and 498 miRNAs from Egg, L3, L3f, L4, L5, male and female, respectively, with 75,95,82,77,79,80, and 85 as known.We further predicted the targets of these miRNAs and performed blast2go analysis of these targets. Gene Ontology terms of miRNA targets are involved in nematode larval development, embryo development, reproduction, molting cycle, collagen and body morphogenesis.The functional characterization of these miRNAs was not known. Accordingly our study evaluated the functional characterization of three miRNAs, miRNA-36, miRNA-100b, miRNA-81a, miRNA mimics, inhibitors and negative controls were designed and their knock-in and knock-down efficiency were analyzed by qRT-PCR after soaping in their specific developmental stages. The functional role of each individual miRNA was analyzed with various parameters of development such as, numbers of larvae collected from the livers and lungs of infected mice 4 days after infection with inhibitor,mimic treated and untreated larvae. The knock-in with the specifically designed miRNA mimics showed increased levels of expression of miRNA compared with their normal controls. We confirmed these findings using qRT-PCR, by in vivo expression in developmental stages of A. suum by soaking method. The knock-down of expression with the corresponding inhibitors showed a considerable decrease in the expression levels of these miRNAs and obvious functional effects in A. suum development. This study carried out by knock-in and knock-down of specifically and temporally expressed miRNAs in A. suum by soaking method is a novel study to delineate the importance of the miRNA expression in regulating A. suum development. For mir-36 and 100b, the knock-down and loss of function of endogenously expressed miRNAs by the miRNA inhibitors in specific developmental stages had considerable effects on development, but enhancement of their gain of function was not observed on knock-in of these specific miRNAs. Hence, our study indicates that an optimal level of endogenous expression of miRNA is indispensable for the normal development, while the knock-in and knock-down of mir-81a had no significant effects on A. suum development. Our findings would facilitate to understand the regulation mechanism of growth and development in A. suum and discover novel targets for anti-parasite drugs regulates or controls.