Identification And Charaterization Of The MiRNA In The Parasitic Famale Adults And Third Stage Larvae In The Parasitic Nematode,Strongyloides Stercoralis

Micro RNAs are a type of non-coding small RNAs with a length of 18-22 nt,which can completely bind to target m RNA to degrade m RNA,or partially bind to m RNA,inhibit the protein translation process of m RNA,and play an important role in the posttranscriptional regulation process.In parasites,miRNAs are being increasingly studied for their potential roles in lifespan,growth and development,infection,host-parasite interactions and pathogenesis through multiple mechanisms.Strongyloides stercoralis is a human and canine gastrointestinal nematode,affecting an estimated 100 million people worldwide.The life cycle of S.stercoralis is unique with obligatory parasitic generation and free-living generation.In healthy individuals,S.stercoralis infection is usually asymptomatic,however,when the host is immunocompromised,the infection may trigger a hyper-infection,causing extremely high mortality,which is a risk factor threating human health worldwide.In the present study,the identification and functional analysis of miRNAs from the third-stage larvae(i L3)and parasitic females(p F)of S.stercoralis were carried out,the findings provide foundation for further functional studies.We obtained the following findings:1.RNA sequencing quality of S.stercoralisTotal RNA of S.stercoralis p F and i L3 larvae was extracted,and high-throughput sequenced with a Solexa sequencer at BGI-Shenzhen,China.s RNA sequencing data showed approximately 0.553 G,0.550 G,0.526 G,0.529 G,0.533 G,0.544 G total bases in the i L3-1,i L3-2,i L3-3,and p F-1,p F-2,p F-3,respectively,and GC content was about 50%,Q20>99%,Q30>94%.The sequencing date were compared with S.stercoralis genome,about 88% and 53% sequences were matched with p F and i L3 sequences,respectively.2.Sequence characteristics of small RNALength distribution in i L3 of S.stercoralis ranged from 18 nt to 30 nt,but most of the clean reads were 22 nt in length.The first base bias analysis of miRNA showed that the first base of miRNA was biased to uracil(U).Non-relevant RNAs including r RNAs,t RNAs,sn RNA,sno RNA,other small RNAs,repeat,exon and intron were removed from the library.After removing these clean sequences,the rest 1.73%of i L3 miRNAs and 18.17%of p F miRNAs which had no match and were marked as unannotated were regarded as miRNA candidates,including known and novel miRNAs.3.Identification of differentially expressed miRNAsWith the blasting search,in both developmental stages,in total 265 miRNAs were identified,including 130 conserved miRNAs and 135 novel miRNAs specific in S.stercoralis being matched to the mature miRNAs of Strongyloides ratti.Among these miRNAs,134 miRNAs were differentially expressed in two libraries of S.stercoralis,including 77 conserved miRNAs and 57 novel miRNAs.More than 21 miRNAs with|Log2Fold Change|>3 have been identified.The hierarchical cluster analysis of differentially expressed miRNAs revealed high correlation between the three biological replicates,verifying the accuracy of data obtained from this experiment.4.Evolutionary analysis of conserved miRNAsThe evolutionary analysis of conserved miRNAs revealed that higher organisms express more miRNAs than lower organisms,and miR-34 and miR-92 are ubiquitous in metazoan.This indicates that the two miRNAs are highly conserved in metazoans and may play important roles in different organisms.5.Target genes analysis between differentially expressed miRNAsBased on the present study results,134 differentially expressed miRNAs were predicated to target 1494 genes using the miRanda.In the molecular function category,the targets of miRNAs are mainly enriched in transmembrane transporter activity,signal transducer activity,nucleic acid binding.In the cellular component category,the targets of miRNAs are mainly enriched in cell and myosin complex.In the biological process category,the targets of miRNAs are mainly enriched in DNA recombination and signal transduction,DNA integration.The KEGG pathways enrichment analysis demonstrated that the target genes perform different functions in the multimedia-signaling pathways,those signaling pathways including AGE-RAGE signaling pathway in diabetic complications,thyroid hormone signaling pathway,Fox O signaling pathway,tight junction,glucagon signaling pathway,micro RNAs in cancer.Further comparative analysis between target genes of S.stercoralis and the homologous genes of other nematodes revealed that those target genes were predicted to play functions of molting,lifespan,regulating motor function,embryonic development and regulating egg laying behavior.6.Validation of miRNAsUsing the cloning method,the pre-miRNAs of S.stercoralis were PCR amplified and cloned into vector,and then were sent to the company for sequencing.It was found that the sequencing results were completely consistent with the deep sequencing,indicating the high quality of the sequencing data.Eleven miRNAs were selected to validate our bioinformational analysis on differential expression between the two developmental stages by stem-loop RT-PCR,and nine miRNAs were differentially expressed,indicating the same trend between RT-RCR data and miRNA-seq.7.Analysis of miRNA expression under stress conditionsRT-PCR was employed to test the expression profiles of ten miRNAs Sst-miR-71-5p,Sst-miR-86-5p,Sst-miR-92-3p,Sst-miR-1-3p,Sst-miR-9-3,Sst-lin4-3p,Sst-miR-81a-5p,Sst-miR-34a-5p,Sst-novel-104,Sst-miR-84-5p under different stress conditions.In temperature stress,at 12℃,37℃,45℃ compared to natural living conditions 22℃,it was found that most miRNAs were down-regulated,of which Sst-novel-104 and Sst-miR-84-5p were up-regulated at 12°C.The results of hypoxia stress showed that Sst-miR-84-5p,Sstlin4-3p,Sst-novel-104,Sst-miR-92-3p were significantly down-regulated,and Sst-miR-81a-5p,Sst-miR-1-3p and Sst-miR-71-5p were significantly up-regulated,while Sst-miR-86-5p,Sst-miR-34a-5p,Sst-miR-9-3p had no significant difference.It was found that all miRNAs were significantly down-regulated under UVC stress.In conclusion,miRNAs in the i L3 and p F of S.stercoralis were characterized in this study.It was found that the differentially expressed miRNAs plays an important role in the movement,regulation of life span,larva development and spawning of S.stercoralis.The stress experiment showed that the changes of miRNA expression under stress conditions suggest that some miRNAs respond to stress,and may be involved in stress response.The results laid a foundation for further study on the functions of miRNA in the S.stercoralis,and provided basis for further screening miRNAs which could be used as drug targets.