Downregulation Of MiR-206 And Its Relationship With FN1 In Mice Infected With H1N1 Swine Influenza

Swine influenza is a highly contagious respiratory disease caused by swine influenza virus,which can cause emphysema,pulmonary edema,pulmonary fibrosis and so on in clinical pathological change.At the same time,the potential hazards of human caused by swine influenza virus,has important significance in public health.Pulmonary fibrosis is characterized by fibroblast proliferation,extracellular matrix with aggregation and inflammation and tissue damage.Fibronetin1 is an important component of extracellular matrix,so is a key protein in pulmonary fibrosis.MicroRNAs is a class of endogenous non-coding RNA,which can regulate the translation of mRNA having been proved to be involved in many life processes,such as embryonic development,tumor,infection and immunity.Studies have shown that these miRNAs target multiple signaling pathways,immune system and inflammatory response,or inhibit influenza infection by direct targeting of influenza virus genes,inhibit influenza virus replication.The mice lung RNA was extracted which has infected highly pathogenic influenza post 5d,and found that miR-206-3p was significantly down regulated by deep sequencing.Bioinformatics analysis suggested that FN1 might be a target gene of miR-206-3p.In this study,we established a mouse model of H1N1 infection to detect the levels of miR-206-3p and FN1 transcription in different stages of the mice lung infected with influenza virus by the method of fluorescence quantitative PCR.At the same time,western-blot was used to detect the expression of FN1 protein in different stages of infected lung.Finally,to verify whether FN1 is the target gene of miR-206-3p by detecting fluorescence signal in wild type and mutant type,luciferase reporter plasmids containing wild type FN1 3'UTR fragment and binding site mutation were transfected into 293 T cells to change the level of miR-206-3p.The results of qRT-PCR detection showed that miR-206-3p of mice infected with H1N1 swine influenza virus 12 h decreased significantly,and with the development of infection,the expression of miR-206-3p reply gradually while the level of FN1 transcription in the infection period did not change significantly.The results of Western-blot showed that the expression of FN1 protein increased in the lung after the infection of H1N1 influenza virus 24 h,and reached the peak after the expression of48 h,and decreased after 72 h.The results of dual luciferase reporter system showed that there was no significant difference between the expression of the wild-type plasmid and the mutant plasmid.The results confirm that FN1 is not a target gene of miR-206-3p,and the upregulation of FN1 protein level is not directly related to miR-206-3p,and it can be speculated that other regulatory factors affect the level of FN1 protein.