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MicroRNA regulation of human protease genes essential for influenza virus replication.

Meliopoulos VA, Andersen LE, Brooks P, Yan X, Bakre A, Coleman JK, Tompkins SM, Tripp RA - PLoS ONE (2012)

Bottom Line: However, the rapid emergence of drug resistance has emphasized the need for new drug targets.The genes validated as critical for influenza virus replication were ADAMTS7, CPE, DPP3, MST1, and PRSS12, and pathway analysis showed these genes were in global host cell pathways governing inflammation (NF-κB), cAMP/calcium signaling (CRE/CREB), and apoptosis.Analyses of host microRNAs predicted to govern expression of these genes showed that eight miRNAs regulated gene expression during virus replication.

View Article: PubMed Central - PubMed

Affiliation: Department of Infectious Diseases, University of Georgia, Athens, Georgia, United States of America.

ABSTRACT
Influenza A virus causes seasonal epidemics and periodic pandemics threatening the health of millions of people each year. Vaccination is an effective strategy for reducing morbidity and mortality, and in the absence of drug resistance, the efficacy of chemoprophylaxis is comparable to that of vaccines. However, the rapid emergence of drug resistance has emphasized the need for new drug targets. Knowledge of the host cell components required for influenza replication has been an area targeted for disease intervention. In this study, the human protease genes required for influenza virus replication were determined and validated using RNA interference approaches. The genes validated as critical for influenza virus replication were ADAMTS7, CPE, DPP3, MST1, and PRSS12, and pathway analysis showed these genes were in global host cell pathways governing inflammation (NF-κB), cAMP/calcium signaling (CRE/CREB), and apoptosis. Analyses of host microRNAs predicted to govern expression of these genes showed that eight miRNAs regulated gene expression during virus replication. These findings identify unique host genes and microRNAs important for influenza replication providing potential new targets for disease intervention strategies.

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Effect of miRNA inhibition on host protease gene expression 24 h post-miRNA inhibitor treatment.Host cell miRNAs of interest were evaluated for their effect on host gene hits by qPCR. A549 cells were treated with the appropriate miRNA inhibitor (25 nM) for 24 hours. Cellular RNA was isolated 24 hpi and evaluated by qPCR for host gene expression using a SYBRgreen assay with gene-specific primers. Gene expression was compared to cells transfected with siNEG (for siRNA) or NEG (non-targeting miRNA inhibitor) at the equivalent concentration. Data is normalized to GAPDH expression. miRNAs indicated on the x-axis refer to inhibition of those miRNAs. A: ADAMTS7 expression levels, B: CPE, C: DPP3, D: MST1, E: PRSS12. Data is representative of two independent experiments. (*p<0.05 versus siRNA treatment.).
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pone-0037169-g005: Effect of miRNA inhibition on host protease gene expression 24 h post-miRNA inhibitor treatment.Host cell miRNAs of interest were evaluated for their effect on host gene hits by qPCR. A549 cells were treated with the appropriate miRNA inhibitor (25 nM) for 24 hours. Cellular RNA was isolated 24 hpi and evaluated by qPCR for host gene expression using a SYBRgreen assay with gene-specific primers. Gene expression was compared to cells transfected with siNEG (for siRNA) or NEG (non-targeting miRNA inhibitor) at the equivalent concentration. Data is normalized to GAPDH expression. miRNAs indicated on the x-axis refer to inhibition of those miRNAs. A: ADAMTS7 expression levels, B: CPE, C: DPP3, D: MST1, E: PRSS12. Data is representative of two independent experiments. (*p<0.05 versus siRNA treatment.).

Mentions: Pathway analysis of the five validated host genes revealed potential miRNA interaction (Figure S4) with eight miRNAs (miR-1254, miR-1272, miR-17-5p, miR-17-3p, miR-106B, miR-106B*, miR-124-a, and miR-124*). To determine the role of these miRNAs in regulating ADAMTS7, CPE, DPP3, MST1, and PRSS12, A549 cells were treated with miRNA hairpin inhibitors as previously described [52], or treated with a negative control from C. elegans that shares no homology with known human miRNA sequences, and gene expression levels determined by qPCR. As gene modulation by miRNAs is usually subtle and multi-targeted, the effect of the miRNA inhibitors on host gene mRNA levels was determined 24 hours post-treatment. All 8 miRNA inhibitors were tested for their effect on each of the 5 validated genes; however, only those inhibitors that affected gene expression are shown (Figure 5). At 24 h post-treatment, ADAMTS7 gene expression levels increased 20-fold when miR-106B was inhibited, and 40-fold when miR-124* was inhibited (Figure 5A). Treatment with siRNA targeting the gene examined was performed as a control. CPE expression was slightly decreased by miRNA inhibitors, as there was no response above 1.0 (Figure 5B). In contrast, inhibition of miR-106B and miR-124* resulted in a >20-fold and >40-fold increase of DPP3 gene expression, respectively (Figure 5C), while miR-1254, miR-1272, and miR-17-3p inhibition caused a decrease of DPP3 expression. MST1 expression was increased to similar levels as DPP3 with the same miRNA inhibitors, while the other miRNA inhibitors resulted in a slight (but significant for miR-17-3p) decrease in expression (Figure 5D). Finally, PRSS12 expression levels were significantly (p<0.05) increased in response to miR-106B inhibition, but a slight decrease was detected by inhibition of miR-1254 (Figure 5E). These results show the same miRNAs can regulate different genes both subtly and robustly.


MicroRNA regulation of human protease genes essential for influenza virus replication.

Meliopoulos VA, Andersen LE, Brooks P, Yan X, Bakre A, Coleman JK, Tompkins SM, Tripp RA - PLoS ONE (2012)

Effect of miRNA inhibition on host protease gene expression 24 h post-miRNA inhibitor treatment.Host cell miRNAs of interest were evaluated for their effect on host gene hits by qPCR. A549 cells were treated with the appropriate miRNA inhibitor (25 nM) for 24 hours. Cellular RNA was isolated 24 hpi and evaluated by qPCR for host gene expression using a SYBRgreen assay with gene-specific primers. Gene expression was compared to cells transfected with siNEG (for siRNA) or NEG (non-targeting miRNA inhibitor) at the equivalent concentration. Data is normalized to GAPDH expression. miRNAs indicated on the x-axis refer to inhibition of those miRNAs. A: ADAMTS7 expression levels, B: CPE, C: DPP3, D: MST1, E: PRSS12. Data is representative of two independent experiments. (*p<0.05 versus siRNA treatment.).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3351457&req=5

pone-0037169-g005: Effect of miRNA inhibition on host protease gene expression 24 h post-miRNA inhibitor treatment.Host cell miRNAs of interest were evaluated for their effect on host gene hits by qPCR. A549 cells were treated with the appropriate miRNA inhibitor (25 nM) for 24 hours. Cellular RNA was isolated 24 hpi and evaluated by qPCR for host gene expression using a SYBRgreen assay with gene-specific primers. Gene expression was compared to cells transfected with siNEG (for siRNA) or NEG (non-targeting miRNA inhibitor) at the equivalent concentration. Data is normalized to GAPDH expression. miRNAs indicated on the x-axis refer to inhibition of those miRNAs. A: ADAMTS7 expression levels, B: CPE, C: DPP3, D: MST1, E: PRSS12. Data is representative of two independent experiments. (*p<0.05 versus siRNA treatment.).
Mentions: Pathway analysis of the five validated host genes revealed potential miRNA interaction (Figure S4) with eight miRNAs (miR-1254, miR-1272, miR-17-5p, miR-17-3p, miR-106B, miR-106B*, miR-124-a, and miR-124*). To determine the role of these miRNAs in regulating ADAMTS7, CPE, DPP3, MST1, and PRSS12, A549 cells were treated with miRNA hairpin inhibitors as previously described [52], or treated with a negative control from C. elegans that shares no homology with known human miRNA sequences, and gene expression levels determined by qPCR. As gene modulation by miRNAs is usually subtle and multi-targeted, the effect of the miRNA inhibitors on host gene mRNA levels was determined 24 hours post-treatment. All 8 miRNA inhibitors were tested for their effect on each of the 5 validated genes; however, only those inhibitors that affected gene expression are shown (Figure 5). At 24 h post-treatment, ADAMTS7 gene expression levels increased 20-fold when miR-106B was inhibited, and 40-fold when miR-124* was inhibited (Figure 5A). Treatment with siRNA targeting the gene examined was performed as a control. CPE expression was slightly decreased by miRNA inhibitors, as there was no response above 1.0 (Figure 5B). In contrast, inhibition of miR-106B and miR-124* resulted in a >20-fold and >40-fold increase of DPP3 gene expression, respectively (Figure 5C), while miR-1254, miR-1272, and miR-17-3p inhibition caused a decrease of DPP3 expression. MST1 expression was increased to similar levels as DPP3 with the same miRNA inhibitors, while the other miRNA inhibitors resulted in a slight (but significant for miR-17-3p) decrease in expression (Figure 5D). Finally, PRSS12 expression levels were significantly (p<0.05) increased in response to miR-106B inhibition, but a slight decrease was detected by inhibition of miR-1254 (Figure 5E). These results show the same miRNAs can regulate different genes both subtly and robustly.

Bottom Line: However, the rapid emergence of drug resistance has emphasized the need for new drug targets.The genes validated as critical for influenza virus replication were ADAMTS7, CPE, DPP3, MST1, and PRSS12, and pathway analysis showed these genes were in global host cell pathways governing inflammation (NF-κB), cAMP/calcium signaling (CRE/CREB), and apoptosis.Analyses of host microRNAs predicted to govern expression of these genes showed that eight miRNAs regulated gene expression during virus replication.

View Article: PubMed Central - PubMed

Affiliation: Department of Infectious Diseases, University of Georgia, Athens, Georgia, United States of America.

ABSTRACT
Influenza A virus causes seasonal epidemics and periodic pandemics threatening the health of millions of people each year. Vaccination is an effective strategy for reducing morbidity and mortality, and in the absence of drug resistance, the efficacy of chemoprophylaxis is comparable to that of vaccines. However, the rapid emergence of drug resistance has emphasized the need for new drug targets. Knowledge of the host cell components required for influenza replication has been an area targeted for disease intervention. In this study, the human protease genes required for influenza virus replication were determined and validated using RNA interference approaches. The genes validated as critical for influenza virus replication were ADAMTS7, CPE, DPP3, MST1, and PRSS12, and pathway analysis showed these genes were in global host cell pathways governing inflammation (NF-κB), cAMP/calcium signaling (CRE/CREB), and apoptosis. Analyses of host microRNAs predicted to govern expression of these genes showed that eight miRNAs regulated gene expression during virus replication. These findings identify unique host genes and microRNAs important for influenza replication providing potential new targets for disease intervention strategies.

Show MeSH
Related in: MedlinePlus