Study On Expression Profiles Of LncRNA And CircRNA In Tracheas Of Chickens Infected With Cryptosporidium Baileyi

Cryptosporidium baileyi,a predominant species infecting poultry,mainly inhabits in the larynx,trachea,bursa of Fabricius,cloaca of hosts,causing respiratory diseases and considerable economic losses to the poultry industry.However,no effective preventive measures and therapeutic drugs were developed against C.baileyi infection.Recent studies have shown that non-coding RNAs were involved in interactions between hosts and pathogens through regulating host immune responses against viruses,bacteria,and parasitic protozoa.Especially,long non-coding RNA(lncRNA)and circular RNA(circRNA)played important regulatory roles in the occurrence and development of many diseases,and could be identified as effective biomarkers for the diagnosis and prognosis of several diseases.Herein,chicken infected with C.baileyi was used as a model to study the expression profiles of host lncRNA,mRNA and circRNA associated with C.baileyi infection for the first time in this study.1.The model of chickens infected with C.baileyi was successfully established:Chickens were orally infected with 1×10~6 C.baileyi oocysts after normal feeding for 3 days.The results showed that the presence of oocysts was detected in the faecal samples from 5 to 29 days post infection(d pi)and the tracheal tissue samples from 2 to 27 d pi.The sequence analysis identified these oocysts as C.baileyi.The oocyst excretion showed that the peaks were presented at 10 and 16 d pi,and the highest amounts of oocysts was observed at 10 d pi.A lot of parasites were detected in the tracheas of the experimental chickens at 10 d pi,but no parasites were seen in the tracheas of the control group with haemotoxylin&eosinstaining and electron microscopy.2.The expression profiles of lncRNA and mRNA in chickens infected with C.baileyi were obtained:The RNA samples of trachea tissues from the chickens infected with C.baileyi at 10 d pi were extracted,and the lncRNA and mRNA were sequenced.A total of 559 574746 raw reads were obtained,and 545 479 934 clean reads were remaining after filtering out the low-quality sequences.After screening,14 698 mRNAs and 1376 new lncRNAs were finally identified,including 1161 long intergenic non-coding RNAs(lincRNAs)and 215antisense lncRNAs.110 lncRNAs(53 up-regulated and 57 down-regulated)and 948 mRNAs(656 up-regulated and 292 down-regulated)were obtained between the experimental and the control groups with the standard of the fold change(FC)>2.0 and q<0.05.Ten mRNAs(five up-and five down-regulated)and ten lncRNAs(five up-and five down-regulated)were randomly selected for qRT-PCR validation.The results of qRT-PCR were consistent with the results of RNA sequencing(RNA-seq)thus verified the validity of RNA-seq analysis.Furthermore,the mRNA levels of partial differentially expressed genes(six mRNAs and six lncRNAs)were detected by qRT-PCR after C.baileyi infection at different times(6 h,12 h,24 h,48 h,72 h,5 d,10 d,16 d,28 d and 29 d pi),and dynamic changes were detected.Target gene prediction showed 634 780 relationships between 14 418 mRNAs and 2570lncRNAs.More than one mRNAs were predicted to be regulated by one lncRNA,and one mRNA corresponded to several lncRNAs.These lncRNAs may be involved in the infection and resolvolution process of C.baileyi by regulating the IL-4,IL-10,IL-17 and other cytokines.Functional enrichment analysis revealed that differentially expressed mRNAs and the target genes of lncRNAs were significantly enriched in immune pathways associated with Cryptosporidium infection.3.The expression profiles of circRNA in chickens infected with C.baileyi were obtained:The tracheal tissues from experimental and control group chickens at 10 d pi were sent for circRNA sequencing,and a total of 9085 circRNAs were obtained.Under the criteria of FC>2.0 and p<0.05,104 differentially significant circRNAs(65 up-and 39 down-regulated)were identified.The qRT-PCR was used to verify six randomly selected circRNAs(three up-and three down-regulated),and the results were consistent with the RNA-seq results.The possible interaction relationships between 9085 circRNAs and 978 miRNAs were observed by the target gene prediction.The source genes of the differentially expressed circRNAs were mainly enriched in some metabolic pathways.In addition,qRT-PCR was performed to analyze the expression levels of four differentially expressed circRNAs(two up-and two down-regulated)and the results showed that the dynamic expressions of these differentially expressed circRNAs were found during C.baileyi infection,suggesting that they would be involved in the interaction between host and pathogen.Collectivelly,the high-throughput sequencing technology was used to examine the expression profiles of mRNA,lncRNA and circRNA of chickens infected with C.baileyi for the first time in this study.A total of 948 mRNAs,110 lncRNAs and 104 circRNAs were found to be differentially expressed,and these RNAs possibly play key roles in the interaction between C.baileyi and host by regulating the expression of their related target genes.Additionally,the signaling pathways associated with these predicted mRNAs,lncRNAs and circRNAs would be used to fully reveal the pathogenesis of C.baileyi infection.