Regulation Of MicroRNA In Lung Inflammatory Injury During The Infection Of Influenza A Virus Subtype H1N1

Influenza viruses are RNA viruses of the family Orthomyxoviridae. They can spread rapidly and cause seasonal flu outbreaks, causing large number of people and animals’ death and huge economic loss. MicroRNAs are short ribonucleic acid(RNA) molecules at the average length of 22bp. They can incorporated into RNA-induced silencing complexes (RISCs) termed miRISC which can functionally target specific RNAs for cleavage or translational repression. Datasets of miRNA microarray and mRNA microarray was compared with the help of functional analysis. MiRNAs’ targets on mice mRNA and H1N1 genome was predicted. The result of targeting verification is useful to understand the function of miRNA in lung inflammatory injury and H1N1 viral replication.The A/Porto Rico/8(PR8) infected mice model was verified in this research. Physical sign of the mice was significantly changed. Average body weight loss begins from day 2 and get to the lowest point at day 9, since then weight gain occurred follow with the recovery of mice. Mortality rate shows that mice die occurred on day 2, and mortality rate of day 5 was the largest, and no mice die after day 10. Q-PCR detection shows that the expression of IL2 was up regulated more than 2 times, and IL10 was nearly 7 times.MiRNA microarray and mRNA microarray of A/Porto Rico/8(PR8) and A/Beijing/501(BJ501) infected mice model were analyzed. In the genes that been chosen BDKRB1, C1QA, C1R, C2, CCL19, CCL3, ITGAM, SERPINE1, THBS1, CXCL11, IL6, FGA, TNFSF14 were up regulated and BMPR2, HC were down regulated. In miRNA chips, there are 47 miRNAs’ expression significantly changed of the two model groups on day 2 and day 5. Up regulated miR-139-5p, miR-466h-5p, miR-130b, miR-21, miR-3470a miR-1187, miR-3082-5p, miR-466i-5p, miR-574-5p; and down regulated miR-1, miR-30d, miR-145, miR-24-2, miR-574-3p were selected. Since the inflammation injury of the two models are different on day 5, miRNA expression between the two model groups were compared. Four miRNAs named mmu-miR-1, mmu-miR-133a, mmu-miR-133b, mmu-miR-449a exprssed higher in group of BJ501 than that of PR8; mmu-miR-1187, mmu-miR-1196, mmu-miR-3082-5p and other nine miRNAs’ expression is lower in BJ501 group than PR8 group.Targetscan, miRbase and other databases were used for prediction of miRNAs’ targets on mRNAs,18 pairs of possible target combinations were found. Among those prediction results miR-1 has the largest number of possible target sequence, named TNFSF14, IL6, THBS1, CXCL11, CCL3, FGA, miR-21 can target on four genes named HC, BMPR2, CXCL11, CCL3. Meanwhile, a miRNA may have a number of different target sites on one mRNA.MiRNA influence viral replication and encoding by targeting viral genome RNAs. RNAhybrid and other forecasting tools were used. MiRNAs of the same expression trend target on conserved sequence of the two virus strains were predicted.8 possible target sets were found, namely miR-468-3p targeting PB2, miR-139-3p, miR-145a-5p targeting HA, miR-139-3p targeting NP, miR-449a-5p target to NA, miR-2137, miR-2861, miR-3082-5p targeting M1 and M2. In addition, NA gene sequences of BJ501 strain has an extra long fragment RNA compared to PR8 strain, causing long fragment amino acids increased in NA protein; mmu-miR-449a can target on this RNA fragment.Fluorescent reporter vectors were constructed. MiRNA target on mRNA were tested by dual luciferase reporter gene system. It shows that miR-574-3p can be targeted on C2 gene 3’UTR, so that the fluorescence intensity of the reporter gene decreased 76.74% compared with the blank group, and that decreased 77.05% compared with the negative control group. Pathway analysis shows that C2 can be involved in inflammation, microbial infections, lung disease, etc.Here, miRNAs’ function by targeting host mRNA in inflammatory injury when H1N1 influenza virus infected were studied, and its function in viral replication by targeting influenza virus genome sequence is also discussed. Those results can be theoretical and experimental basis for the development of anti-influenza drugs.