摘要
AIM: To test whether the replication of human rhinovirus(HRV) is regulated by micro RNAs in human bronchial epithelial cells.METHODS: For the present study, the human cell line BEAS-2B(derived from normal human bronchial epithelial cells) was adopted. DICER knock-down, by si RNA transfection in BEAS-2B cells, was performed in order to inhibit micro RNA maturation globally. Alternatively, antisense oligonucleotides(anti-mi Rs) were transfectedto inhibit the activity of specific micro RNAs. Cells were infected with HRV-1B. Viral replication was assessed by measuring the genomic viral RNA by reverse transcription quantitative polymerase chain reaction(RT-q PCR). Association between micro RNA-induced-silencing-complex and viral RNA was detected by Ago2 co-immunoprecipitation followed by RT-q PCR. Targetscan v.6 was used to predict micro RNA target sites on several HRV strains.RESULTS: Here, we show that micro RNAs affect replication of HRV-1B. DICER knock-down significantly reduced the expression of mature micro RNAs in a bronchial epithelial cell line(BEAS-2B) and in turn, increased the synthesis of HRV-1B RNA. Additionally, HRV-1B RNA co-immunoprecipitated with argonaute 2 protein, an important effector for micro RNA activity suggesting that micro RNAs bind to viral RNA during infection. In order to identify specific micro RNAs involved in this interaction, we employed bioinformatics analysis, and selected a group of micro RNAs that have been reported to be under-expressed in asthmatic bronchial epithelial cells and were predicted to target different strains of rhinoviruses(HRV-1B,-16,-14,-27). Our results suggest that, out of this group of micro RNAs, mi R-128 and mi R-155 contribute to the innate defense against HRV-1B: transfection of specific anti-mi Rs increased viral replication, as anticipated in-silico.CONCLUSION: Taken together, our results suggest that pathological changes in micro RNA expression, as already reported for asthma or chronic obstructive pulmonary disease have the potential to affect Rhinovirus replication and therefore may play a role in virusinduced exacerbations.
AIM: To test whether the replication of human rhinovirus(HRV) is regulated by micro RNAs in human bronchial epithelial cells.METHODS: For the present study, the human cell line BEAS-2B(derived from normal human bronchial epithelial cells) was adopted. DICER knock-down, by si RNA transfection in BEAS-2B cells, was performed in order to inhibit micro RNA maturation globally. Alternatively, antisense oligonucleotides(anti-mi Rs) were transfectedto inhibit the activity of specific micro RNAs. Cells were infected with HRV-1B. Viral replication was assessed by measuring the genomic viral RNA by reverse transcription quantitative polymerase chain reaction(RT-q PCR). Association between micro RNA-induced-silencing-complex and viral RNA was detected by Ago2 co-immunoprecipitation followed by RT-q PCR. Targetscan v.6 was used to predict micro RNA target sites on several HRV strains.RESULTS: Here, we show that micro RNAs affect replication of HRV-1B. DICER knock-down significantly reduced the expression of mature micro RNAs in a bronchial epithelial cell line(BEAS-2B) and in turn, increased the synthesis of HRV-1B RNA. Additionally, HRV-1B RNA co-immunoprecipitated with argonaute 2 protein, an important effector for micro RNA activity suggesting that micro RNAs bind to viral RNA during infection. In order to identify specific micro RNAs involved in this interaction, we employed bioinformatics analysis, and selected a group of micro RNAs that have been reported to be under-expressed in asthmatic bronchial epithelial cells and were predicted to target different strains of rhinoviruses(HRV-1B,-16,-14,-27). Our results suggest that, out of this group of micro RNAs, mi R-128 and mi R-155 contribute to the innate defense against HRV-1B: transfection of specific anti-mi Rs increased viral replication, as anticipated in-silico.CONCLUSION: Taken together, our results suggest that pathological changes in micro RNA expression, as already reported for asthma or chronic obstructive pulmonary disease have the potential to affect Rhinovirus replication and therefore may play a role in virusinduced exacerbations.
基金
Supported by MRC,AAIR and the Roger Brooke charitable trust
