Screening Of Differientially Expressed CircRNAs And Their CeRNA Network In The Liver Of Mice Infected With Echinococcus Multilocularis Larvae

Circular RNAs(circRNAs)are a kind of covalently closed circular RNAs produced by back-splicing.They are abundant in eukaryotes,evolutionarily conservative,tissue-specific,highly stable,and accumulates with aging in neural tissues.CircRNAs can be cis-regulated by competing for splicing with their corresponding linear RNA products.Echinococcus larval infection is commonly known as echinococcosis or hydatidosis.Hydatidosis(hydatid disease)is an important zoonosis caused by the larvae of Echinococcus tapeworms parasitizing the liver,lungs and other tissues and organs of humans and animals.Alveolar echinococcosis(AE)is one of mankind’s most deadly zoonoses,which is caused by E.multilocularis(Em)larvae and is a widely distributed in the northern hemisphere.Also,it is a kind of natural epidemic disease.About 17,400 cases of the infection are annually reported,most of which occur in China.Echinococcus multilocularis larvae primarily occur within the host liver,from which the larvae can shift to other organs such as lungs,brains,etc.and even can spread from the surface of the organs into the body cavity,like a malignant tumor,so also known as "worm cancer".The harm of echinococcosis to the host mainly consists of the capture of the host nutrients,the mechanical damage to the host and the effects of toxicity and antigenicity,thus causing a chronic immune responses and inflammation in the host without obvious clinical symptoms.As a result,patients with E.multilocularis larvae cannot be easily detected in the early stage of infection.The specific or differential expression of circRNAs in liver tissues of mice infected with E.multilocularis larvae in the early stage was investigated to further understand the changing trend of circRNAs in the host post infection,to explore their feasibility of the new stable and sensitive molecular biomarkers used for diagnosis and prognosis,to illuminate their function and molecular interaction mechanisms,and to improve the levels of diagnosis and treatment,which could lay a preliminary foundation and provide a baseline for future research.Main contents and results in this study are as follows:1.The differentially expressed circRNA microarray was analyzed using high-throughput sequencing techniques for the liver tissues of infected and uninfected mice.According to bioinformatics analysis,differentially expressed circRNAs were screened out.Four up-regulated circRNAs and two downregulated circRNAs were selected according to the principle of high abundance and large differentially expressed circRNA.qRT-PCR(quantitative real-time fluorescence PCR)was used to verify whether the expression trend was consistent with the high-throughput sequencing results.Sequencing results showed that 201 circRNA genes were up-regulated and 9 circRNA genes were down-regulated,with a total of 210 differentially expressed circRNA genes.qRT-PCR verified that the trend of 4 up-regulated and that of 2 down-regulated circRNA genes were consistent with the chip results.Six differentially expressed circRNAs were verified,which proved that the high-throughput sequencing results were reliable.Sanger sequencing was used to confirm that the splice sites of two circRNAs were suitable.2.Bioinformatics analysis was conducted on the differentially screened circRNAs,and their circRNA-miRNA(micro RNAs)-mRNA interplay networks were constructed to display putative molecular mechanisms of function and action of the circRNAs.GO enrichment analysis showed that they enriched molecular function,biological process and cell components.KEGG enrichment pathway analysis revealed that these circRNAs were involved in endocytosis,SNARE protein-related vesicle movement,mineral absorption and other immune-related signaling pathways.Based on the verified results of microarray qRT-PCR,bioinformatics methods were further used to analyze the possible miRNA binding of the differentially expressed circRNAs in the liver of mice infected with E.multilocularis larvae and the control group,and to investigate the possible pathogenesis of the larvae in the host from the perspective of circRNA regulation.What’s more,circRNA-miRNA-mRNA networks of five candidate circRNAs were designed,which would provide an crucial clues for further investigating their related functions in mice infected with E.multilocularis larvae.In summary,in this study,high-throughput sequencing of circRNAs in hosts infected with E.multilocularis was conducted for the first time,and the changes of circRNAs in the early stage of infection were preliminarily explored.The chip sequencing results were verified to be reliable,the shear sites of two circRNAs were identified,the target circRNAs were analyzed by ceRNAs,and the possible basedon ceRNA molecular interaction networks and mechanisms of five circRNA candidates were analyzed and discussed,which laid a preliminary foundation and provided a baseline for further research on the role and molecular mechanism of circRNAs in the pathogenesis of echinococcosis.