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. 2012 Oct;16(10):2539-46.
doi: 10.1111/j.1582-4934.2012.01572.x.

Cellular microRNA let-7c inhibits M1 protein expression of the H1N1 influenza A virus in infected human lung epithelial cells

Yong-Jie Ma  1 , Jing Yang, Xing-Liang Fan, Hai-Bao Zhao, Wei Hu, Zhen-Peng Li, Guang-Chuang Yu, Xiao-Ran Ding, Jun-Zhi Wang, Xiao-Chen Bo, Xiao-Fei Zheng, Zhe Zhou, Sheng-Qi Wang
Affiliations

Cellular microRNA let-7c inhibits M1 protein expression of the H1N1 influenza A virus in infected human lung epithelial cells

Yong-Jie Ma et al. J Cell Mol Med. 2012 Oct.

Abstract

The influenza virus (IV) triggers a series of signalling events inside host cells and induces complex cellular responses. Studies have suggested that host factors play an essential role in IV replication. MicroRNAs (miRNAs) represent a class of small non-coding RNAs that target mRNAs, triggering either translation repression or RNA degradation. Emerging research suggests that host-derived cellular miRNAs are involved in mediating the host-IV interaction. Using miRNA microarrays, we identified several miRNAs aberrantly expressed in IV-infected human lung epithelial cells (A549). Specifically, miR-let-7c was highly up-regulated in IV-infected A549 cells. PITA and miRanda database screening indicated that the let-7c seed sequence is a perfect complementary sequence match to the 3' untranslated region (UTR) of viral gene M1 (+) cRNA, but not to PB2 and PA. As detected by a luciferase reporter system, let-7c directly targeted the 3'-UTR of M1 (+) cRNA, but not PB2 and PA. To experimentally identify the function of cellular let-7c, precursor let-7c was transfected into A549 cells. Let-7c down-regulated IV M1 expression at both the (+) cRNA and protein levels. Furthermore, transfection with a let-7c inhibitor enhanced the expression of M1. Therefore, let-7c may reduce IV replication by degrading M1 (+) cRNA. This is the first report indicating that cellular miRNA regulates IV replication through the degradation of viral gene (+) cRNA by matching the 3'-UTR of the viral cRNA. These findings suggest that let-7c plays a role in protecting host cells from the virus in addition to its known cellular functions.

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Figures

Fig 1
Fig 1
Cell viability after miRNA expression vector transfection in A549 cells. A549 cells were transfected with different miRNA expression vectors and influenza A/JingFang/86-1(H1N1) virus was inoculated 24 hrs later. Twenty-four hours after the inoculation, cell viabilities were assessed by the CCK-8 assay. Data are expressed as mean ± S.E., n = 4, *P < 0.05.
Fig 2
Fig 2
Viral titres in the supernatants of A549 cells after transfection of miRNA expression vectors. A549 cells were transfected with different miRNA expression vectors and influenza A/JingFang/86-1(H1N1) virus was inoculated 24 hrs later. Viral titres were assessed 48 hrs after inoculation. Data are expressed as mean ± S.E., n = 4, *P < 0.05.
Fig 3
Fig 3
Quantitative real-time PCR analysis of let-7c in influenza virus-infected A549 cells. RNA was isolated from A549 cells after infection with Influenza A/JingFang/86-1(H1N1) virus (A) or influenza A/FM/1/47 (H1N1) virus (B) for 4 or 24 hrs. Let-7c was detected and presented relative to GAPDH mRNA. Data are expressed as mean ± S.E., n = 3, *P < 0.05, #P < 0.01.
Fig 4
Fig 4
M1 vRNA expression and influenza A virus titres after let-7c overexpression in A549 cells. Twenty-four hours after transfection with let-7c expression vector, A549 cells were infected with influenza A/JingFang/86-1 (H1N1) virus or influenza A/FM/1/47 (H1N1) virus. (A) Influenza A/JingFang/86-1 (H1N1) virus titres in the medium of A549 cells were detected at the indicated time points. (B) Influenza A/FM/1/47 (H1N1) virus titres in the medium of A549 cells were detected at the indicated time points. (C) M1 and NP vRNA levels were determined at 4, 6 and 24 hrs after influenza A/JingFang/86-1 (H1N1) virus infection by q-PCR. (D) M1 and NP vRNA levels were determined at 4, 6 and 24 hrs after influenza A/FM/1/47 (H1N1) virus infection by q-PCR. Data are expressed as mean ± S.E., n = 4, *P < 0.01.
Fig 5
Fig 5
Let-7c target site prediction in the vRNA of influenza virus A Let-7c was predicted to pair with residues in the 3′ region of cRNA of M1, PA and PB2 by PITA and miRanda database screens.
Fig 6
Fig 6
Let-7c target analysis using luciferase reporter assays in A549 cells Luciferase reporter assays of A549 cells 48 hrs after cotransfection of the influenza virus M1, PA, PB1 and PB2 3′-UTR reporter constructs and let-7c expression vector. Data were normalized to β-galactosidase activity. Data are expressed as mean ± S.E., n = 3, *P < 0.05.
Fig 7
Fig 7
M1 expression after let-7c overexpression in A549 cells. (A) A549 cells were infected with influenza A/FM/1/47 (H1N1) virus for 24 or 48 hrs. Immunoblot analysis for M1 and NP protein in isolated A549 cell protein homogenate was performed. (B) Twenty-four hours after transfection with let-7c expression vector or with a let-7c inhibitor, A549 cells were infected with Influenza A/JingFang/86-1(H1N1) virus for 24 or 48 hrs. Immunoblot analysis for M1 and NP protein in isolated A549 cell protein homogenate was performed. Data shown are representative of three independent experiments.
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