Screening Study On Differential Expression Of Non-coding RNA In Cytomegalovirus-infected Mouse Cochlear Epithelial Cells

Purpose: Hearing impairment is one of the most common diseases which affect human health.The cytomegalovirus is a very common pathogen that causes congenital infections in infants and young children,and it is also the main cause of non-hereditary deafness in humans.In recent years,with the rapid development of high-throughput sequencing and bioinformatics,a series of non-coding RNAs have entered the vision of researchers.Though studies have shown that cytomegalovirus infection can cause changes in non-coding RNA expression,here is no report on the differential expression of noncoding RNA in the process of deafness caused by cytomegalovirus infection.This study intends to construct the cytomegalovirus-infected mouse cochlear sensory epithelial cell model,and use high-throughput detection technology to detect the expression of non-coding RNA in cochlear sensory epithelial cells infected with cytomegalovirus for 72 hours happening.The basic characteristics of various non-coding RNAs were analyzed by bioinformatics technology,the non-coding RNAs that differentially expressed were screened out and verified by the qPCR method.This study will explore differentially expressed non-coding RNAs that associated with cytomegalovirus-infected-deafness,set up a foundation for further study which detects the mechanism of cytomegalovirus-infected-deafness in transcription and non-coding RNA,and it provide a new direction for further research.Research method:1.Culture of mouse cochlear epithelial cells(MCEC)and mouse embryonic fibroblasts(Balb/3T3).2.Preparation of mouse cytomegalovirus strain(Smith strain)and TCID50 to determine the titer of the virus,and the growth curve of the virus in mouse embryo fibroblasts was draw.3.Construction of the mouse cytomegalovirus-infected mouse cochlear epithelial cell model.4.Apply high-throughput sequencing technology for whole transcriptome sequencing to detect the expression of non-coding RNA in cochlear epithelial cells infected with and without cytomegalovirus for 72 hours.Screen out significantly different non-coding RNAs(miRNAs,lncRNA)and Deafness related genes.5.The non-coding RNAs with significant differential expression and deafness-related genes were verified by the qPCR and analyzed by bioinformatics.Result:1.We found a large number of differentially expressed non-coding RNAs between cytomegalovirus-infected and uninfected mouse cochlear sensory epithelial cells,including 143 miRNAs,of which 62 were newly discovered;3402 lncRNAs,of which 792 were newly discovered;5115 differentially expressed mRNAs were also found.They are mainly involved in ion binding,regulation of genetic material metabolic processes,signal transduction,and regulation of cellular processes.They are mainly involved in signal pathways such as viral infection and the immune system.2.We have screened and verified that there are 10 miRNAs consistent with the trend of sequencing results,of which 4 are newly discovered;2 lncRNAs,all of which are known;2 deafness-related genes-Myo7 a and Tmie,and 3 miRNAs target these two genes.3.The regulatory relationship between Myo7a–(miR-1929-5p),Myo7a-(egr-miR-4990),and Tmie-(miR-6952-3p)was initially obtained by using biochemical analysis and qPCR verification.One lncRNA was predicted to participate in the ceRNA regulatory network of Myo7 a,and 14 lncRNA was involved in the ceRNA regulatory network of Tmie.Conclusion:1.In this study,it was confirmed that in the process of Cytomegalovirus infected cochlear sensory epithelial cells,many non-coding RNAs were differentially expressed.The differentially expressed 143 miRNAs and 3402 lncRNAs were obtained by sequencing.These ncRNAs play a role in the process of deafness caused by cytomegalovirus infection which mainly through exercising biological functions such as ion binding,regulation of the metabolic process of genetic material and signal transduction,participating in signaling pathways such as immune system and virus infection.2.After screening and validation,potential regulatory non-coding RNAs associated with Cytomegalovirus-infected deafness and deafness-related genes-Myo7 a and Tmie were obtained.By bioassay and preliminary verification,the regulatory relationships of Myo7a-(mir-1929-5p),Myo7a-(egr-mir-4990)and Tmie-(mir-6952-3p)were identified,and the ceRNA regulatory network involved in the processing of these two deaf-related genes was predicted.It provides a new research entry point for the mechanism of cytomegalovirus infection causing deafness.