The Mechanism Of MicroRNA-7 Regulating Rotavirus NSP5 And Host IGF1 To Inhibit Virus Replication

Rotavirus(RV)which belongs to Rotavirus genus of reovirus family is composed of 11 RNA segments in genome.The 6 structural proteins(VP 1-4,6,7)and 6 non-structural proteins(NSP1-6),encoded by RV,play an important role in the adsorption,invasion,replication,assembly and release of RV infection process.RV infection can induce abnormal expression in host cells,including miRNAs.As a non-coding small RNA,its regulatory mechanism involved in the growth and development,metabolism,disease occurrence,pathogen infection and other fields of the tissues and organs has always been a focus of research and discussion.In the preliminary experiment,we analyzed the miRNA expression profile of the RV-infected MA104 cells and found that the expression level of miR-7 was significantly up-regulated.So we list miR-7 as the research object to explore its regulation mechanism in RV replication.Objective:this thesis aims to explore the function,the target genes,the regulation mechanism,the involved signaling pathways and other aspects of miR-7 in the process of RV infection.Methods:After the confirmation of miR-7 as the research object,we first detected the effect of miR-7 on RV replication by overexpression/inhibition in vitro(MA104 cells)and in vivo(Balb/c neonatal mice model).For virus genes,we confirmed that the NSP5 gene of RV was a miR-7 target gene through database analysis and prediction and double luciferase reporter assay.Besides,the further verification of miR-7 and NSP5 was conducted by using pEGFP-N2-NSP5 overexpression vector and and mir-7 mimics.For host genes,we also screened another new target gene of miR-7,insulin-like growth factor 1(IGF1),with the some method of NSP5,and detected the effect of overexpression of IGF1 on RV replication at protein level.In addition,the regulation mechanism of IGF1 involved in MAPK(P38,ERK,JNK)and its downstream signaling molecule TGF-? were also detected by RT-qPCR.Results:The expression of miR-7 in host cells was significantly up-regulated after RV infection with different genotypes while the expression of mir-7 was up-regulated with the increase of MOI after RV infection with the same genotype.Results from vitro(MA104 cells)and vivo(Balb/c neonatal mice model)showed that mirR-7 could inhibit RV replication.After the confirmation of RV NSP5 as the target gene of miR-7,we also detected the expression level of NSP5 from the overexpression vector of pEGFP-N2-NSP5 or RV in host cells with up-regulation of miR-7.And the results could further confirm that mir-7 can down-regulate the intracellular RV replication by targeting and inhibiting the expression of NSP5 protein.In addition,the IGF1 in host cell which can regulate RV replication by activating MAPK(P38,ERK,JNK)and downstream target signal molecule TGF-?,is also a new target gene of miR-7.Conclusion:The expression of miR-7 is significantly up-regulated in RV-infected host cells and can be involved in the antiviral mechanism of host cells.Besides,miR-7 can regulate RV replication by targeting and down-regulating the expression of RV NSP5 and intracellular IGF1 protein,which can reduce the number of viroplasms and enhance the intracellular innate immunity that may be inhibited by the activated MAPK signaling pathway,respectively.The miR-7 with its target genes and signaling pathways can provide a theoretical basis for the prevention and treatment of RV infection.