The Expression And Bioinformatics Analysis Of Circular RNAs In Endometritis Mice Uteru Tissues

Circular RNAs（circ RNAs）are covalently closed circular RNAs produced by reverse splicing,which have the characteristics of tissue-specific expression and high stability,and have been confirmed to be involved in the occurrence of various diseases.Endometritis is an inflammation of endometrium caused by pathogen infection.The disease is a common postpartum disease of dairy cows,which seriously affects the reproductive performance of dairy cows and causes huge economic losses in dairy farming.Early diagnosis and treatment can avoid further development of the disease.Escherichia coli（E.coli）is one of the main pathogens of cow endometritis,and it is also the earliest invasive pathogen.This paper aims to study the change trend of circ RNAs after early infection of E.coli in mouse uterus,and analyze it by bioinformatics to explore its potential function and molecular mechanism in endometritis,so as to provide theoretical basis for the clinical diagnosis and treatment of endometritis.In this study,the uterus of mice in the experimental group were perfused with 25 u L of E.coli with different concentrations(1×10⁶、10⁸、10¹⁰CFU/m L).Similarly,the control group was given the same amount of normal saline.q RT-PCR,ELISA,HE staining and Western Blot were used to detect the effects of E.coli on the inflammatory response of mouse endometrium.Then Illumina Hi Seq technology was used to detect the significant differential expression of circ RNAs in the uterus samples of mice in the E.coli treatment group(1×10¹⁰CFU/m L)and the control group,and bioinformatics software was used for functional analysis of the differential expression of circ RNAs.Finally,q RT-PCR and Western Blot were used to verify the differentially expressed circ RNAs and the related genes and proteins of the signal pathway.The main research results are as follows:1.Compared with the control group,the endometrial integrity of mice in 1×10¹⁰ CFU/m L E.coli treatment group was destroyed,the number of glands decreased,the cell spacing increased,and inflammatory cell infiltration was observed.E.coli significantly increased the gene and protein expression levels of inflammatory factors IL-6,IL-1βand TNF-αin mouse uterus,and significantly increased the phosphorylation levels of IκBαand P65.2.Compared with the control group,200 differentially expressed circ RNAs were found in the uterine tissues of mice in the treatment group,of which 83 were significantly up-regulated and 117 were significantly down-regulated.According to the principle of high abundance and large difference multiple,five differentially expressed circ RNAs were screened.q RT-PCR was used to verify that the expression trend was consistent with the sequencing results.Biological software was used to predict the mi RNAs bound to these five circ RNAs.It was found that there was a complex regulatory network between circ RNAs and mi RNAs.3.The GO（Gene Ontology,GO）function results of the differentially expressed circ RNAs parent genes showed that the differentially expressed genes were mainly related to the positive regulation of RNA polymerase II（POLⅡ）promoter transcription,the negative regulation of cell proliferation,cytoplasm,DNA and RNA binding processes.KEGG（Kyoto Encyclopedia of Genes and Genomes,KEGG）signaling pathway analysis showed that differentially expressed genes were mainly related to the endocytosis,TGF-βsignaling pathway,and N-glycan biosynthesis pathway.4.q RT-PCR results showed that E.coli treatment significantly increased the m RNA levels of TGF-β1 and Smad7,but had no significant effect on the m RNA level of Smad3.Western Bolt results showed that E.coli significantly increased the protein level of Smad7 and decreased the phosphorylation level of Smad3.In summary,this study for the first time used high-throughput sequencing technology to analyze circ RNAs in mouse uterus infected with Escherichia coli,to determine the changes of circ RNAs in the early stage of infection,and to verify the screening of five significant differentially expressed circ RNAs to establish their mi RNA network,and to explore the target molecules of their role.At the same time,it is proved that TGF-β/Smad signaling pathway is involved in the occurrence and development of endometritis,which provides reference data for further research.