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. 2015 Dec;240(12):1630-9.
doi: 10.1177/1535370215589051. Epub 2015 Jun 15.

Human miR-3145 inhibits influenza A viruses replication by targeting and silencing viral PB1 gene

Kritsada Khongnomnan  1 , Jarika Makkoch  1 , Witthaya Poomipak  2 , Yong Poovorawan  3 , Sunchai Payungporn  4
Affiliations

Human miR-3145 inhibits influenza A viruses replication by targeting and silencing viral PB1 gene

Kritsada Khongnomnan et al. Exp Biol Med (Maywood). 2015 Dec.

Abstract

MicroRNAs (miRNAs) play an important role in the regulation of gene expression and are involved in many cellular processes including inhibition of viral replication in infected cells. In this study, three subtypes of influenza A viruses (pH1N1, H5N1 and H3N2) were analyzed to identify candidate human miRNAs targeting and silencing viral genes expression. Candidate human miRNAs were predicted by miRBase and RNAhybrid based on minimum free energy (MFE) and hybridization patterns between human miRNAs and viral target genes. In silico analysis presented 76 miRNAs targeting influenza A viruses, including 70 miRNAs that targeted specific subtypes (21 for pH1N1, 27 for H5N1 and 22 for H3N2) and 6 miRNAs (miR-216b, miR-3145, miR-3682, miR-4513, miR-4753 and miR-5693) that targeted multiple subtypes of influenza A viruses. Interestingly, miR-3145 is the only candidate miRNA targeting all three subtypes of influenza A viruses. The miR-3145 targets to PB1 encoding polymerase basic protein 1, which is the main component of the viral polymerase complex. The silencing effect of miR-3145 was validated by 3'-UTR reporter assay and inhibition of influenza viral replication in A549 cells. In 3'-UTR reporter assay, results revealed that miR-3145 triggered significant reduction of the luciferase activity. Moreover, expression of viral PB1 genes was also inhibited considerably (P value < 0.05) in viral infected cells expressing mimic miR-3145. In conclusion, this study demonstrated that human miR-3145 triggered silencing of viral PB1 genes and lead to inhibition of multiple subtypes of influenza viral replication. Therefore, hsa-miR-3145 might be useful for alternative treatment of influenza A viruses in the future.

Keywords: influenza A virus; inhibition; miR-3145; miRNA; silencing; viral replication.

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Figures

Figure 1
Figure 1
Venn-Euler diagram shows a summary of candidate human miRNAs targeting influenza A viruses subtype pH1N1, H5N1, and H3N2. Bullets refer to different types of hybridization patterns between miRNAs and target viral RNA. The prediction showed that 76 human miRNAs target influenza A viruses, including 70 miRNAs that targeted specific subtype (21 for pH1N1, 27 for H5N1 and 22 for H3N2) and 6 miRNAs (miR-216b, miR-3145, miR-3682, miR-4513, miR-4753 and miR-5693) that targeted multiple subtypes of influenza A virus
Figure 2
Figure 2
The alignment of viral PB1 gene revealed conserve target sites for seed region of hsa-miR-3145. Various strains in representative of four subtypes (pH1N1, H3N2, H5N1 and H7N9) of influenza A virus infecting humans were included in the alignment. The alignment was performed using Clastal W implemented in BioEdit software
Figure 3
Figure 3
The bar graph shows the relative luciferase activity among groups of transfected cells. Group 1, cells were transfected with pGL3MS2/Basic and pSilencer_scramble as the control group; group 2, cells were transfected with pGL3MS2/Basic and pSilencer_FLuc was used as the positive silencing control; group 3, cells were transfected with pGL3MS2/Basic and pSilencer_miR-3145 in order to test whether miR-3145 triggered non-specific complementary with a firefly luciferase gene; group 4, cells were transfected with pGL3MS2/Basic_PB1 and pSilencer_scramble to test a silencing effect triggered by base complementation between an endogenous cellular miR-3145 and the PB1 gene within 3′-UTR of the firefly luciferase gene; group 5, cells were transfected with pGL3MS2/Basic_PB1 and pSilencer_FLuc as another positive silencing control; group 6, cells were transfected with pGL3MS2/Basic_PB1 and pSilencer_miR-3145 to test the silencing effect of miR-3145. The pCMV_RLuc control vector was transfected into cells in each group. ‘*’ refers to significant differences from controls. Results revealed that the relative luciferase in groups 2, 5, and 6 were significantly lower (P < 0.05) than controls
Figure 4
Figure 4
Percentage of relative expression of viral PB1 gene normalized with GAPDH gene quantitated by real-time PCR. The result demonstrated the significant reduction (P < 0.05) of viral PB1 gene in the presence of miRNA-3145 compared to mock infection in all three subtypes of influenza A virus including pH1N1, H5N1 and H3N2. ‘*’ refers to significant differences from the control (mock infection)
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