Selection And Functional Analysis Of MIRNA Involved In Injury Of Inflammation Induced By A Influenza Virus (H1N1)

Influenza is acute contagious respiratory disease caused by influenza virus, andhas the characters of wide spread, rapid onset and great harm. A large number ofstudies have shown that lung inflammatory injury was mediated by the influenza virus,which is an important reason to cause death. After pathological studies of severe casesand animal experiments, shows that the2009H1N1virus has the ability to lead tosevere pneumonia directly. Previous studies have proved that the serious degree oflungs inflammation leaded by Influenza virus is closed with the host complementactivation and the viral induction―Cytokine Storm‖. miRNA is playing the importantregulative role in pneumonia occurrence developing process, but the Immunitymechanism of virus pneumoniais is not clear, particulaly, miRNA play the role in theprocess which has not been in deeply study. Therefore, we established mouse modelrespectively using two kinds of strains (BJ501and PR8)of H1N1influenza Avirus.The host mRNA regulating mechanism of lung inflammatory damage by fluvirus using the miRNA chip and mRNA chip is discussed and it is further revealingthe pathogenic mechanism of influenza virus.First, we established mouse pneumonia model of flu virus using influenza Avirus (strain A/Puerto Rico/8/1934H1N1)(PR8)and (strain A/Beijing/501/2009)(BJ501). Through observation of signs and detection of weight, lung index, pathology,cytokine, Complement molecule,adhesion molecule,we found the model were in linewith the flu-like symptoms, on the seventh day, the weight dropped to a minimum, thelung index of PR8group achieved to peak on the seventh day, BJ501group achievedto peak on the ninth day. and appearing pathology phenotype of obvious pneumonia,the expression of mediators of inflammation obviously up-regulation.On the fifth daywe concluded that the PR8group was significantly superior to BJ501group.Theconclusion was based on the following finding:the degree of weight loss,the lungindex increase,tissue lesions found and the expression level of theC3aR,IL6,VCAM-1, IFN-γ,TNF-α.Our finding suggests that we have successfullyestablished mouse pneumonia models for these two strains of influenza.The lunginflammatory injury caused by the PR8strain was slightly more severe than BJ501 strain.We used the miRNA chip to examine miRNA expression of model mouse lungtissue infected by flu virus on the2ndand5thday.The analysis of miRNA expressionshows that10increased miRNAs and none decreased were detected on the secondday among PR8group through comparing with control group. On the5thday, therewere42changed miRNAs were detected, in which36miRNAs were increased, and6miRNAs were decreased. Among BJ501group, there were6changed miRNAs weredetected on the second day, in which4miRNAs were increased, and2miRNAs weredecreased, there were25changed miRNAs were detected on the fifth day, in which22miRNAs were increased, and3miRNAs were decreased. Through comparison oftwo model groups and PBS group, we found that the specific change of miRNAs is46and40among PB8group and BJ501group respectively, in which30miRNAs in PR8group and BJ501group occurred a significant change, of which28miRNAs raised,two down, mainly including of miR-155and of miR-130b, miR-21, miR-2137,miR-1949, miR-468,miR-574-3p, miR-145, miR-24-2*.Previous studies have shownthat miR-155, miR-468, miR-145, miR-24-2involved in immune regulation and playa role in the inflammatory response process. In our experiment, miR-468expressionsin the two groups is higher than BJ501group at the same time, which suggesting thatmiR-468is a reason of PR8group was more inflammation than BJ501group at thesame time.Our results show that the number and expression of miRNAs was changed withthe pneumonia increase. MiRNA changes is different when mice were infected bydifferent flu virus strains. The results also proved that host miRNA expressionchanges involved in influenza virus-mediated immune injury of the lunginflammation.The analysis of mRNA expression shows that106increased mRNAs and120decreased were detected on the second day among PR8group through comparing withcontrol group. On the5thday, there were1999changed mRNAs were detected, inwhich1439mRNAs were increased, and560mRNAs were decreased. Among BJ501group, there were226changed mRNAs were detected on the second day, in which106miRNAs were increased, and120miRNAs were decreased, there were1730changed mRNAs were detected on the fifth day, in which1177mRNAs were increased, and553mRNAs were decreased.Further analysis revealed that afterinfection, cytokines, complement, and adhesion molecules such as: IL6, CXCL11,CXCL10and TNF, IFN, C3aR, C5aR, CD62P and VCAM-1, etc,which wereoccurred in significant changes in lung tissue. The results with our animal model ofthe process of establishing IL6, TNF-alpha and IFN-γ, C3aR, C5aR, CD62P andVCAM-1PCR results are consistent. At the protein level, CD62P and C4d wereverified entirely consistent with the microarray results by Western-blot method. Theresults show that the expression profiles of host mRNA has the occurrence ofsignificant changes among different virus strains, and cytokines, complement, andadhesion molecules are involved in the lungs of influenza-mediated immune injury.The analysis of Pathway and GO showed that significantly changed genes inlung tissue was mainly involved in cellular processes, the regulation of cellularmetabolism, immune processes and other aspects. In the PR8group,the more targetgenes involved in the regulation of inflammation-related pathways.Using perl code and Sanger and TargetScan databases, target gene analysis wascarried by unifies miRNA and the mRNA data according to the negative regulativeprinciple. The result discovered the PR8group and the BJ501group separately had24and21miRNA examine the target gene in the mRNA expression spectrum, including16miRNA were shared in the two groups, They were miR-24-2*, miR-155, miR-145,miR-21, miR-223, miR-130a,130b, miR-139-3p, miR-139-5p, miR-18a, miR-7a,et al.Special miRNA in PR8group were miR-30d, miR-133a, miR-133b, miR-142-5p,miR-680, miR-877*, miR-1, miR-20*. Special miRNA in BJ501group weremiR-142-3p, miR-574-3p, miR-126-5p, miR-125a-3p, miR-671-5p.We also discovered that gene is related with the inflammation correlation cellsignal passage by futher analysis. Some miRNA inhibited the cell signaling pathways,some miRNA activate the signaling pathways, Such as miR-223, miR-130b,miR-24-2*activated apoptosis signaling pathway, MiR-155inhibited the pathway;miR-145inhibit the activation of the complement and coagulation cascades pathway,miR-24-2*activated pathway, miR-574-3p activated the complement pathway theninhibited it. In addition, miR-30d in the PR8group sustained activated C1R toactivate complement pathway, but activated SERPINE1is inhibition of thecomplement pathway, it also activat the gene of SOCS-3, which was negative to the adjustment of IL-6. This reflects the diversity of the regulation of miRNA to pathwayand suggests a balance state in host-virus interaction. The results show that the samemiRNA in the same signaling pathway can also play opposite role.In the Cytokine-of cytokine receptor Signaling pathway, PR8group had moremiRNAs (miR-30d, miR-1, miR-133a, miR-133b) activated IL6,IL21R, TNF,CXCL11and other genes, the signaling pathway was activated through comparedwith the BJ501group. The result shows that differences of miRNAs was the reason tothe degree of inflammation in PR8group and BJ501group.The result of gene chip showed miR-574-3p in the BJ501group activating theC5aR on the5th day, which consistent with real RT-PCR results. It indicates thatmiR-574-3p is perhaps one of the reasons for the C5aR in the BJ501group was higherexpressed than in the PBS group.MiRNA is the upstream modifier of inflammation members.Penetrates toresearch the inflammation response aspect is certainly helpful to the thoroughexposition flu virus result pneumonia immunity mechanism,and may guide the newmedicine the development and the application.