Identification Of MiRNAs Encoded By BCG And Its Role In BCG Growth

[Objective]MicroRNAs(miRNAs)are a class of small non-coding RNAs that post-transcriptionally regulate gene expression.miRNAs are widely found in plant,mammalian,drosophila melanogaster,C.elegans,and viruses.However,no evidence indicates that miRNAs also exist in prokaryotes.The aim of this study was to identify whether mycobacterium encodes noncoding small RNA and to explore their function on growth and its biogenesis mechanism.[Methods]First,the total RNA was isolated from mycobacterium.Small RNAs are purified for Solexa sequence,which collect all of total RNA fragments' sequence.Mireap was perfoimed to predict second structure of small RNA.Northern Blot was carried out to identify that these predicted miRNAs are exist in BCG.Real-time PCR was applied for exploring endogenous expression level.Then endogenous miRNA was blocked or over-expressed by electroporation for observing its role of growth in BCG.Finally,a fluorescence report system was performed to confirm that whether candidates,which are predicted by bioinformatics software,are real target gene.[Results]In this study,the Solexa sequencing approach was used to sequence a small RNA library prepared from Mycobacterium bovis BCG str.Pasteur 1173P2,and 388 known miRNAs and 42 novel miRNAs were identified using a bioinformatics approach and some of them were confirmed by Northern Blot.One of them,BCG-miR-07 can affect growth and drug resistance of M.tb,which down-regulate the expression of ICD2 and ABC transporter by bioinformatics prediction and Fluorescence report experimental.[Conclusions]Our results confirmed that miRNAs exist in BCG for the first time.These results indicate BCG-miR-07 and BCG-miR-21 inhibit growth of BCG.This is also first evidence that miRNAs exist in Prokaryotes.