MicroRNA Regulation Of RIG-I Like Receptor Signaling Pathway And Analysis Of MicroRNA Function In Miiuy Croaker

Innate immunity serves as the first line of defense against pathogens,which play a critical role in recognizing viral infections and become a hot research area.A series of receptors were identified and their functions were extensive researched.Retinoic acid induced gene I(RIG-I)like receptors can sending viral nucleic acids and active the downstream genes in RIG-Isignaling pathway.To date,many researches focus on the immune response,the regulation of immune response has been little investigated.In recent years,microRNA(miRNA)were indicated that play key regulatory roles in diverse biological processes via RNA silencing and post-transcriptional regulation of gene expression.In order to comprehend the regulatory mechanism of miRNAs,two small RNA libraries from the spleen of miiuy croaker individuals with or without poly(I:C)infection were constructed.The characteristic of miRNA and the regulation of miRNA to immune response by targeting the genes form RIG-I signaling pathway.Further,analyzing the regulation of miR-145 on its target gene MDA5 are betterfor understanding the functions of miRNA.we obtained some main conclusions:1.The Illumina deep-sequencing platform were used to sequencing,and then two sRNA libraries were obtained.All tags align to Rfam database 12,052,153 and 11,150,433 clean reads were obtained from the control and infected libraries,respectively.The clean reads were aligned to miRBase database,and total 219 and 217 number of miRNAs were identified in the two libraries,respectively.Evaluating the hairpin structures of the non-redundant sRNAs,total 75 and 69 unique novel miRNAs were identified from the two libraries of miiuy croaker,respectively.2.Total of 14 conserved and 8 novel differentially expression miRNAs were identified.After poly(I:C)stimulation 9 conserved miRNAs and 2 novel miRNAs had higher expression.After poly(I:C)stimulation 5 conserved miRNAs and 6 novel miRNAs had lower expression.Four differently expression miRNAs(miR-132-3p,miR-155,miR-181 a and miR-190a)of miiuy croaker were selected to confirm the expression pattern of miRNAs by quantitative stem-loop RT-PCR analyses.The result implied that high throughput sequencing method is feasible.3.In this study,the regulation of miRNAs to RIG-I signaling pathway in miiuy croaker was regarded as the model to better understand the functions of miRNAs in the immune response.Many genes from RIG-I signaling pathway were identified as the target gene of differential expression miRNAs,such as NLRX1,JNK,IRF3 and CASP8.The expression pattern of miRNA and their target gene were researched for better understand the negative regulation of miRNA.All the studies will be useful for understanding the regulation of miRNA to immune response.RIG-I like receptors,RLRs family contain three members: RIG-I,melanoma differentiation associated gene 5(MDA5)and laboratory of genetics and physiology2(LGP2).The gene RIG-I were lost in many fish and the LGP2 can't active the downstream genes due to lost the CARD domain.Therefore,MDA5 play a significant role in immune response and the researches about the regulation to MDA5 are significant.In this study,the MDA5 was identified the putative target gene of miR-145 by bio-informatics.Then the target sites from the 3'untranslated region of MDA5 transcripts were confirmed by using luciferase reporter assays.Pre-miR-145 was more effective in inhibiting MDA5 than miR-145-5pmimic,and the effect was dose-and time-dependent.The expression patterns of miR-145-5p and its target gene MDA5 were analyzed inmiiuy croaker liver and kidney tissues.Results implied that miR-145-5p mayfunction via degrading the MDA5 mRNAs,thereby regulating the RIG-I signaling pathway.Studies on miR-145-5p will enrich knowledge of its functions in immune response regulation in fish,as well as offer abasis for regulatory networks that are composed of numerous miRNAs.