Study On The Mechanism Of MiR-142a Family Affecting Inflammatory Response During Bacterial Infection In Ctenopharyngodon Idellus

Grass carp(Ctenopharyngodon idella)is a kind of herbivorous freshwater fish,which is rich in nutrition and has high economic value.In recent years,due to the intensive cultivation of C.idella,the breeding environment has gradually deteriorated,leading to the occurrence of diseases,in which sepsis is one of the most common diseases of C.idella.Septicemia of C.idella is a systemic inflammatory response syndrome caused by Aeromonas hydrophila infection,which causes high mortality and economic losses.Lnc RNA and mi RNA play an important role in the regulation of inflammatory response.In this paper,we used A.hydrophila as pathogen model to analyze lnc RNA(lnc RNA-WAS,lnc RNA-C8807)and mi RNA(mi R-142a-5p)of C.idella.The results are as follows:1.Previous studies have shown that mi R-142a-3p can regulate the expression of downstream inflammatory factors by targeting tnfap2 and glut3.In this study,RNAhybrid was used to predict the lnc RNA binding to mi R-142a-3p of C.idella.According to the prediction results,we selected two lnc RNAs,named lnc RNA-WAS and lnc RNA-C8807 respectively,and verified them by sequencing.It was found that the expression levels of lnc RNA-WAS and lnc RNA-C8807 in CIK infected by Aeromonas hydrophila increased at first,then decreased,and finally increased,which was negatively correlated with the expression pattern of mi R-142a-3p.Overexpression of lnc RNA-WAS and lnc RNA-C8807 could significantly inhibit the expression of mi R-142a-3p.The dual luciferase reporter gene system confirmed that lnc RNA-WAS and lnc RNA-C8807 could directly bind to mi R-142a-3p.Transfection of lnc RNA-WAS or lnc RNA-C8807 overexpression vector can significantly improve the activity of NF-κB and the expression level of downstream inflammatory factors.2.Based on the identification of 16 highly expressed mi RNAs in the spleen of C.idella disease resistant population in the early stage,the subsequent functional analysis of mi R-142a-5p was carried out.The potential target genes of mi R-142a-5p,par2 and tbk1,were predicted by RNAhybrid.During the infection of CIK cells by A.hydrophila,the expression levels of par2 and tbk1 increased first,then decreased,and finally increased,contrary to the expression pattern of mi R-142a-5p.Overexpression or inhibition of mi R-142a-5p can significantly inhibit or increase the expression of par2 and tbk1.The dual luciferase reporter gene system further confirmed the targeted regulation of mi R-142a-5p on par2 and tbk1.Mi R-142a-5p regulates the expression of tnf-α,il-6 and il-1β by targeting par2 and tbk1.3.The c DNA sequence of tnfaip2 was obtained and identified.The results of q PCR showed that tnfaip2 was expressed in different degrees in all tested C.idella tissues.The expression level of tnfaip2 was higher in gill,head kidney and kidney,and lower in brain and muscle.After infection with A.hydrophila,there was a significant immune response to tnfaip2 in all four immune tissues of C.idella.During the infection of CIK cells by A.hydrophila,the expression of tnfaip2 changed significantly.After transfection with the over expression vector of tnfaip2,the expression level of downstream inflammatory factors was significantly increased.