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. 2012;7(1):e30030.
doi: 10.1371/journal.pone.0030030. Epub 2012 Jan 17.

MicroRNA-221 modulates RSV replication in human bronchial epithelium by targeting NGF expression

Sreekumar Othumpangat  1 , Cheryl Walton, Giovanni Piedimonte
Affiliations

MicroRNA-221 modulates RSV replication in human bronchial epithelium by targeting NGF expression

Sreekumar Othumpangat et al. PLoS One. 2012.

Abstract

Background: Early-life infection by respiratory syncytial virus (RSV) is associated with aberrant expression of the prototypical neurotrophin nerve growth factor (NGF) and its cognate receptors in human bronchial epithelium. However, the chain of events leading to this outcome, and its functional implications for the progression of the viral infection, has not been elucidated. This study sought to test the hypothesis that RSV infection modulates neurotrophic pathways in human airways by silencing the expression of specific microRNAs (miRNAs), and that this effect favors viral growth by interfering with programmed death of infected cells.

Methodology: Human bronchial epithelial cells infected with green fluorescent protein-expressing RSV (rgRSV) were screened with multiplex qPCR arrays, and miRNAs significantly affected by the virus were analyzed for homology with mRNAs encoding neurotrophic factors or receptors. Mimic sequences of selected miRNAs were transfected into non-infected bronchial cells to confirm the role of each of them in regulating neurotrophins expression at the gene and protein level, and to study their influence on cell cycle and viral replication.

Principal findings: RSV caused downregulation of 24 miRNAs and upregulation of 2 (p<0.01). Homology analysis of microarray data revealed that 6 of those miRNAs exhibited a high degree of complementarity to NGF and/or one of its cognate receptors TrKA and p75(NTR). Among the selected miRNAs, miR-221 was significantly downregulated by RSV and its transfection in bronchial epithelial cells maximally inhibited gene and protein expression of NGF and TrKA, increased apoptotic cell death, and reduced viral replication and infectivity.

Conclusions/significance: Our data suggest that RSV upregulates the NGF-TrKA axis in human airways by silencing miR-221 expression, and this favors viral replication by interfering with the apoptotic death of infected cells. Consequently, the targeted delivery of exogenous miRNAs to the airways may provide a new strategy for future antiviral therapies based on RNA interference.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. RSV-induced change in microRNAs expression.
This heat map was generated using differentially expressed genes based on unadjusted t-test p-values, and provides an overall picture of the impact of RSV infection on the human bronchial epithelial cell miRNAome as compared to non-infected control cells. Data were partitioned between RSV and control (CTL) samples during the hierarchical clustering and organized into blocks of columns. The rows represent wells in each of the plates, and within each row the green shaded areas indicate lower expression of the specific miRNA noted on the right, whereas the red shaded areas indicate higher expression. Black and darkly shaded areas indicate relatively similar expression between infected and non-infected cells. The Euclidean clustering method was used for array data analysis, and the cutoff level of statistical significance was set at p<0.01 to reduce clutter. The expression index is shown in the right lower corner.
Figure 2
Figure 2. Gene expression in bronchial cells transfected with microRNAs.
Homo sapiens (hsa)-miRNAs with a high degree of homology to mRNAs encoding neurotrophic factors or receptors were selected from our microarray dataset, and mimic oligonucleotides of the precursors for each of these miRNAs were transfected into human bronchial epithelial cells to study their individual role in modulating neurotrophin expression. PCR measured maximally reduced concentrations of both NGF and TrKA transcripts in bronchial epithelial cells overexpressing miR-221. Data are expressed as the mean ± SEM (n = 3–4 per group). * = p<0.01 compared to cells transfected with the negative control miRNA.
Figure 3
Figure 3. Flow cytometry of bronchial cells transfected with microRNAs.
Consistent with the gene expression data, miR-221 transfection was associated to maximal downregulation of NGF protein synthesis, and was also associated to maximal downregulation of its high-affinity TrKA receptor. Data are expressed as the mean ± SEM. Geometric mean fluorescent intensity (MFI) was averaged from the flow cytometry measurements obtained in 3 or 4 independent experiments. * = p<0.01 compared to cells transfected with the negative control miRNA [control(-ve)].
Figure 4
Figure 4. Programmed death of RSV-infected cells.
(A) Non-infected cells transfected with hsa-miR-221 were more prone to apoptosis detected by annexin V staining compared to cells transfected with the negative control miRNA. In contrast, hsa-miR-221 transfection did not change the proportion of necrotic cells detected by propidium iodide (PI) staining. (B) After infection with rgRSV, approximately 3 times more hsa-miR-221-transfected cells became apoptotic compared to cells transfected with negative control miRNA. Left upper quadrant = necrotic cells; left lower quadrant = viable cells; right lower quadrant = apoptotic cells; right upper quadrant = late apoptotic cells in necrotic state.
Figure 5
Figure 5. Combined effect of miR-221 and RSV on NGF expression.
(A) Flow cytometry analysis of hsa-miR-221-overexpressing bronchial epithelial cells 24 h after infection with rgRSV showed significantly lower expression of NGF protein compared to infected cells with normal miR-221 expression. The gray population represents the isotype control. (B) Geometric mean fluorescent intensity (MFI) was averaged from the flow cytometry measurements obtained in 3 or 4 independent experiments. Data are expressed as the mean ± SEM. * = p<0.01 compared to cells transfected with the negative control miRNA.
Figure 6
Figure 6. Confocal microscopy of RSV-infected cells.
After 24 h infection with rgRSV, most of the bronchial epithelial cells transfected with negative control miRNA (top panels) showed strong green fluorescence as a result of active rgRSV infection and concomitant red fluorescence upregulation of NGF expression. In contrast, NGF expression was silenced in bronchial epithelial cells overexpressing hsa-miR-221 (bottom panels), and this effect was associated with a sharp reduction in the number of cells displaying green fluorescence. Bar scale = 20 µm.
Figure 7
Figure 7. Anti-viral effect of miR-221.
(A) FACS showed that hsa-miR-221 transfection reduces significantly the number of rgRSV-infected bronchial cells compared to control cells with normal hsa-miR-221 expression. Geometric mean fluorescent intensity (MFI) was averaged from the flow cytometry measurements obtained in 3 independent experiments. (B) Even stronger inhibition was measured in separate experiments by the titration of fluorescent virus infectious units per ml (IU/ml; n = 6 per group). Data are expressed as the mean ± SEM. ** = p<0.01 compared to cells transfected with the negative control miRNA.
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