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Non-coding RNAs are a class of RNAs that generally do not code for any protein. Different types of ncRNAs are present in mammalian cells including miRNA and lncRNAs. miRNAs are small ncRNA containing about 22 nucleotides. They regulate post-transcriptional gene expression by a mechanism that is known as "RNA interference". lncRNAs are relatively large RNAs with a length of more than 200 nucleotides and have various functions such as carrying out both gene inhibition and gene activation through diverse mechanisms. miRNAs have been shown to have a role in mycobacterial pathogenesis; however, little is known about lncRNAs in mycobacterial infection. Mycobacteria are intracellular pathogens that are hard to eradicate because of their ability to inhibit phagolysosome formation. Approximately onethird of the global population is affected by mycobacterial infections. Nothing was known about involvement of lncRNAs and miRNAs in phagosomal maturation upon mycobacterial infection. In the present study, following infection of monocyte derived macrophages with BCG, expression of lncRNAs as well as miRNAs were studied at different time points of incubation. Time points for RNA isolation were selected based on two aspects: Colocalisation studies of the phagosomal marker protein LAMP1 with BCG, and cascades of TLR4 signalling, which occurs in the initial period of mycobacterial infection. Differences in LAMP1 localisation on the phagosomes of both groups were observed at 30 min and 4 h.
In the first part of the work, after establishing a lncRNA-based RT-qPCR protocol, several preselected lncRNAs that had functions related to immunity were observed to be downregulated at 30 min in BCG infected macrophages compared to non-infected cells as well as heat-killed controls. Among all of them, MEG3 was a highly downregulated lncRNA. In silico analysis predicted that MEG3 had functions in mTOR and PI3K-AKT signalling pathways suggesting its role in autophagy. Induction of autophagy with IFN-γ in infected macrophages resulted in sustained MEG3 downregulation and lack of IFN-γ allowed for counter-regulation of MEG3 by viable BCG. Knockdown of MEG3 in macrophages resulted in induction of autophagy and enhanced eradication of intracellular BCG.
In the second part of the work, a bottom up approach was used to identify miRNAs and respective targets that are involved in phagosomal maturation processes in mycobacterial infection. In silico analysis sorted most enriched miRNAs, and expression studies identified downregulated miRNAs (miR-3619-5p, miR-637 and miR-324-3p) at different time points in BCG infected cells (irrespective of viable or HK BCG) compared to non-infected cells. After identifying their targets, expression studies showed upregulation of lysosomal cysteine protease Cathepsin S (CTSS) and Rab11 family-interacting protein 4 (RAB11FIP4). Reporter gene assays verified regulation of CTSS by miR-3619-5p. Finally, downregulated miR-3619-5p affected autophagy in macrophages.
In conclusion, the present study has identified a role of lncRNA MEG3 in mycobacterial infection, as well as that of miRNA miR-3619-5p along with its target CTSS. Both ncRNAs had functions in autophagy-related pathways during mycobacterial infection.