Screening Of Differential MicroRNAs Of Trichinella Spiralis After Invasion Of Host Intestinal Epithelial Cells And Its Interaction With Target Genes

Backgrounds and AimsTrichinellosis is a serious parasitic zoonosis mainly caused by eating raw or undercooked pork and the meat of other animals containing Trichinella spiralis larvae.At present,trichinellosis is still very popular in Russia and Eastern European countries,Mexico,Chile,Argentina,Thailand and other places.hundreds of trichinosis outbreaks have occurred in many provinces or areas in China.It is estimated that the current number of trichinella infections is more than 20 million.Symptoms such as nausea,vomiting,fatigue,hypothermia,abdominal pain,diarrhea or constipation may be associated with trichinella infection,and severe patients may be accompanied by persistent high fever,eyelid and facial edema,systemic myalgia,myocarditis,pneumonia or encephalitis.microRNA?miRNA?is an endogenous non-coding single-stranded RNA of about 19 to 24 nucleotides long,which could cause degradation or translation inhibition of the target mRNA by specific binding to the target mRNA.At present,it has been found that miRNA expression in a variety of parasites is specific,such as sja-miR-1 and sja-miR-7-5p of Schistosoma japonicum are related to larval maturation,sja-mi R-36-3p is associated with the initial development of schistosomulum.The miR-146a-5p,miR-132a-3p,which are involved in pathogenesis of Angiostrongylus cantonensis,may be associated with the regulation of the genes in the inflammation of the central nervous system.There are few studies on miRNAs of Trichinella spiralis,which are mainly concerned with miRNAs involved in the development and maturation of trichinella in the physiological state,and the key miRNAs associated with pathogenesis are rarely reported.In this study,Solexa high throughput sequencing was used to screen the differentially expressed miRNAs of Trichinella spiralis after invasion of host intestinal epithelial cells.Bioinformatics analysis was used to predict the target genes of differentially expressed miRNAs to investigate the molecular regulation mechanism in the process of invasion for the prevention and treatment of trichinellosis and screening of new preventive molecular targets.The following studies were carried out:?1?RNA was extracted from Trichinella spiralis muscle larvae and intestinal infective larvae and subjected to high throughput sequencing.Real time PCR was used to validate the sequencing results.?2?Select the specific miRNA,predict the target genes by RNAhybrid and TargetScan,and verify the relationship between the specific miRNA and its target genes by dual luciferase report gene assay.Real time PCR was used to detect the expression levels of specific miRNA and its target genes to provide a basis for revealing the effect of the specific miRNA and its target genes in the regulation of Trichinella spiralis invasion of host intestinal epithelial cells.Methods1 In this study,artificial digestion was used to collect Trichinella spiralis muscle larvae,and the activated muscle larvae was cultured with HCT-8 cells to collect the intestinal infective larvae,and the RNA of the two groups were extracted for high throughput sequencing.Real time PCR was used to detect the expression levels of some randomly selected miRNAs.2 The target genes of the specific miRNA were predicted by RNAhybrid,TargetScan,MiRanda and PicTar,and the recombinant plasmids were constructed and transfected into HCT-8 cells with miRNA mimics to detect the expression of the reporter gene,thereby verifying the relationship between specific miRNA and its target genes.Real time PCR was used to detect the expression levels of specific miRNA and its target genes.Results1 The results of high-throughput sequencing showed that 3431 conservative miRNAs were differentially expressed in the Trichinella spiralis intestinal infective larvae,in which 1466 miRNAs were up-regulated and 1965 miRNAs were down-regulated.In addition,78 new miRNAs were also predicted.Eighteen miRNAs were randomly selected from the sequencing results and detected by real time PCR.The results were consistent with the sequencing data.2 In consideration of the expression level,gene sequence,biological activity prediction,regulation of target gene function,and miRNA binding to its target genes,the relationship between Tsp-let-7 and its four possible genes?Tsp₀6966,Tsp₀7700,Tsp₀7369,Tsp₁4768?was detected by dual luciferase report gene assay.The results showed that Tsp₀6966 and Tsp₀7700 were the potential target genes regulated by Tsp-let-7,and real time PCR showed that the expression level of Tsp-let-7 in intestinal infective larvae was significantly decreased,while the expression of Tsp₀6966 and Tsp₀7700 were increased,which suggested that Tsp-let-7 might negatively regulate the expression of Tsp₀6966 and Tsp₀7700 to participate in the pathogenesis of Trichinella spiralis invasion of host intestinal epithelial cells.Conclusions1 There do exist differentially expressed miRNAs of Trichinella spiralis after invasion of host intestinal epithelial cells.2 Tsp₀6966 and Tsp₀7700 are the potential target genes on which Tsp-let-7 directly acted.3 Tsp-let-7 may negatively regulate the expression of Tsp₀6966 and Tsp₀7700 to participate in the pathogenesis of Trichinella spiralis invasion.