BackgroundMany efforts have been made to understand basal mechanisms of mycobacterial infections. Macrophages are the first line of host immune defence to encounter and eradicate mycobacteria. Pathogenic species have evolved different mechanisms to evade host response, e.g. by influencing macrophage apoptotic pathways. However, the underlying molecular regulation is not fully understood. A new layer of eukaryotic regulation of gene expression is constituted by microRNAs. Therefore, we present a comprehensive study for identification of these key regulators and their targets in the context of host macrophage response to mycobacterial infections.Methodology/Principal FindingsWe performed microRNA as well as mRNA expression analysis of human monocyte derived macrophages infected with several Mycobacterium avium hominissuis strains by means of microarrays as well as quantitative reverse transcription PCR (qRT-PCR). The data revealed the ability of all strains to inhibit apoptosis by transcriptional regulation of BCL2 family members. Accordingly, at 48 h after infection macrophages infected with all M. avium strains showed significantly decreased caspase 3 and 7 activities compared to the controls. Expression of let-7e, miR-29a and miR-886-5p were increased in response to mycobacterial infection at 48 h. The integrated analysis of microRNA and mRNA expression as well as target prediction pointed out regulative networks identifying caspase 3 and 7 as potential targets of let-7e and miR-29a, respectively. Consecutive reporter assays verified the regulation of caspase 3 and 7 by these microRNAs.Conclusions/SignificanceWe show for the first time that mycobacterial infection of human macrophages causes a specific microRNA response. We furthermore outlined a regulatory network of potential interactions between microRNAs and mRNAs. This study provides a theoretical concept for unveiling how distinct mycobacteria could manipulate host cell response. In addition, functional relevance was confirmed by uncovering the control of major caspases 3 and 7 by let-7e and miR-29a, respectively.
Background: MicroRNAs (miRNAs) are small endogenous non-coding interfering RNA molecules regarded as major regulators in eukaryotic gene expression. Different methods are employed for miRNA expression profiling. For a better understanding of their role in essential biological processes, convenient methods for differential miRNA expression analysis are required.
Tumour necrosis factor-α (TNF-α) is a double-edged cytokine associated with pathogenesis of inflammatory-related cancers being also able to induce cancer cell death. In the process of tumour development or metastasis, cancer cells can become resistant to TNF-α. In trefoil factor 3 (TFF3) overexpressing colorectal adenocarcinoma cells (HT-29/B6), we observed enhanced resistance against TNF-α/interferon gamma-induced apoptosis. TFF3 is a secreted small peptide that supports intestinal tissue repair but is also involved in intestinal tumour progression and scattering. We hypothesised that TFF3 rescues intestinal epithelial cancer cells from TNF-α-induced apoptosis by involving regulatory RNA networks. In silico-based expression analysis revealed TFF3-mediated regulation of selected microRNAs as well as long non-coding RNAs (lncRNAs), whereas miR-491-5p was identified to target the lncRNA ‘psoriasis susceptibility-related RNA gene induced by stress’ (PRINS). RNA interference-based gain- and loss-of-function experiments examined miR-491-PRINS axis to exert the TFF3-mediated phenotype. Chemical inhibition of selected pathways showed that phosphatidylinositol 3-kinase/AKT accounts for TFF3-mediated downregulation of miR-491-5p and accumulation of PRINS. Moreover, we showed that PRINS colocalises with PMAIP1 (NOXA) in nuclei of HT-29/B6 possessing inhibitory effects. Immunoprecipitation experiments proved molecular interaction of PMAIP1 with PRINS. Our study provides an insight into RNA regulatory networks that determine resistance of colorectal cancer cells to apoptosis.
bProbiotics are widely used in human and animal health, but little is known about the mode of action of probiotics. One possible mechanism at the molecular level could be an influence on microRNAs (miRNAs) and the related immune-relevant target genes. Here, we analyzed differential expression of miRNA and potential target genes of ileal and jejunal lymphatic tissues from Enterococcus faecium NCIMB 10415-fed piglets versus untreated controls by using next-generation sequencing. We identified miR-423-5p as being greatly affected by the treatment group (2.32-fold; P ؍ 0.
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