Abstract
MicroRNAs (miRNAs) have been well known to play diverse roles in viral infection at the level of posttranscriptional repression. However, much less is understood about the mechanism by which miRNAs are regulated during viral infection. It is likely that both host and virus contain factors to modulate miRNA expression. Here we report the up-regulation of microRNA-15b (miR-15b) in vitro upon infection with Japanese encephalitis virus (JEV). Analysis of miR-15b precursor, pri-miR-15b and pre-miR-15b, suggest that the regulation occurs transcriptionally. Further, we identified the transcriptional regulatory region of miR-15b that contains consensus binding motif for NF-κB subunit c-Rel and cAMP-response element binding protein (CREB), which are known as transcription factor to regulate gene expression. By promoter fusion and mutational analyses, we demonstrated that c-Rel and CREB bind directly to the promoter elements of miR-15b, which are responsible for miR-15b transcription in response to JEV infection. Finally, we showed that pharmacological inhibition of ERK and NF-κB signaling pathway blocked induction of miR-15b in JEV infection, suggesting important roles of ERK and NF-κB pathway in the regulation of miR-15b gene. Therefore, our observations indicate that induced expression of miR-15b is modulated by c-Rel and CREB in response to JEV infection.
Highlights
MicroRNAs are approximately 22 nucleotides evolutionarily conserved non-coding small RNAs1
HeLa cells were transfected with plasmids expressing each of the Japanese encephalitis virus (JEV) proteins. Quantitative real-time PCR (qRT-PCR) analysis showed that exogenous expression of JEV proteins can not alter the levels of miR-15b (Fig. 1d)
These results indicated that up-regulation of miR-15b in JEV infected HeLa cells may rely on viral replication
Summary
MiR-15b is widely expressed and plays diverse roles in different tissues and cell types. Vesicular stomatitis virus (VSV) infection induces miR-146a expression in mouse macrophage in a TRL/MyD88-independent and RIG-I/NF-κ B-dependent manner, and miR-155 expression is upregulated in response to RIG-I activation[39,54] In line with these observations, our data demonstrate that miR-15b induction is dependent on RIG-I since induction of miR-15b by JEV challenge was impaired and binding of c-Rel and CREB to the miR-15b promoter were decreased in RIG-I-knockdown cells. Our results show that miR-15b is induced in HeLa cells after infection with JEV, and the upregulation of miR-15b is transcriptionally regulated These findings suggest that c-Rel and CREB bind to the miR15b promoter and modulate miR-15b transcription. We found that JEV infection upregulates miR-15b expression through RIG-I-dependent manner, and ERK and NF-κ B pathways are involved in miR-15b induction
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