Prediction And Identification Of MiRNA Targeting Toll-like Receptor7in Chicken

Toll-like receptors (TLRs) are fundamental sensor molecules of the host innateimmune system. As a main antiviral receptor in the innate immune system, TLR7mediated antiviral signaling pathways has been always a hotspot. miRNA as animportant mechanism of transcriptional gene regulation, is extensively involved inthe various host life processes. This study is focused on whether miRNA couldregulate the expression of TLR7in the innate immune after Avian influenza virusinvading the body. Firstly, We detected the differences of TLR7expression betweenAvian influenza virus infected chicken and SPF chicken and proved that TLR7genesparticipated in the antiviral mechanism of the innate immune system. Then Wepredicted that a number of miRNAs regulated TLR7expression when Avian influenzavirus chicken’s immune system. By Using bioinformatics methods, we forecastedthese miRNAs were miR107-3p and miR27b-3p. To testify this prediction, weconstructed a dual luciferase reporter gene system to identify the relationship betweenTLR7and the two miRNAs. The results showed that,TLR7expressed both in Avian influenza virus infected chicken and SPF chicken,and the expression was increased obviously in Avian influenza virus infected chicken.1. The results of qPCR showed that the expression of TLR7in immune organ wasupregulated1.7times in spleen,1.65times in bursa of Fabricius and6.7times inthymus, but in the dysfunctional organs, TLR7expression was upregulated4.7timesin lung,1.6times in superior trachea and1.8times in inferior trachea and thediscrepancy was significant.2. Successfully obtained chicken TLR73`UTR sequences by3`RACEexperiments and predicted the3`UTR sequences of the possible role of miRNAtargets by bioinformatics analysis. 3. Successfully predicted the targeting miRNA of TLR7By miRanda, RITAsoftware and Targets can website, and there are targets On3`UTR sequence of TLR7,while RNA chip results and quantitative results also show that these two miRNA mayregulate the relationship betweenTLR7.4. Successfully constructed the dual luciferase reporter vector of miR107-3p,miR27b-3p of WT (wild type), Mut (mutant).5. Optimized co-transfection conditions of mimics (miRNA mimics) withrecombinant plasmid.6. The difference was significant (P<0.01) after the co-transfected of dualluciferase reporter vector of wild-type(WT)and mutant(Mut) with mimics (miRNAmimics), that is wild-type(WT) fluorescence much less than the mutation type(mut)which described that miRNA regulate the expression of TLR7.The research verified that TLR7take part in the immunological mechanism of theinfluenza virus invasion through researching the different expression of TLR7inAvian influenza virus infected chicken and SPF chicken, successfully obtainedmiRNA regulated TLR7gene through analysis and prediction by bioinformatics, thendetected and identified the regulation on TLR7of miRNA. This study providesimportant reference on further comprehension of signal pathway of innate immune ofvirus invading body and the regulation mechanism of miRNA in innate immune, alsoestablishes foundation on studying the role of TLR7in Avian flu infecting poultry.