Intestinal Salmonella typhimurium infection leads to miR-29a induced caveolin 2 regulation.

BACKGROUND: Salmonella are able to modulate host cell functions facilitating both uptake and resistance to cellular host defence mechanisms. While interactions between bacterial modulators and cellular proteins have been the main focus of Salmonella research, relatively little is known about mammalian gene regulation in response to Salmonella infection. A major class of mammalian gene modulators consists of microRNAs. For our study we examined interactions of microRNAs and regulated mRNAs in mammalian intestinal Salmonella infections using a piglet model. METHODOLOGY/PRINCIPAL FINDINGS: After performing microRNA as well as mRNA specific microarray analysis of ileal samples from Salmonella infected as well as control piglets, we integrated expression analysis with target prediction identifying microRNAs that mainly regulate focal adhesion as well as actin cytoskeleton pathways. Particular attention was given to miR-29a, which was involved in most interactions including Caveolin 2. RT-qPCR experiments verified up-regulation of miR-29a after infection while its predicted target Caveolin 2 was significantly down-regulated as examined by transcript and protein detection. Reporter gene assays as well as RNAi experiments confirmed Caveolin 2 to be a miR-29a target. Knock-down of Caveolin 2 in intestinal epithelial cells resulted in retarded proliferation as well as increased bacterial uptake. In addition, our experiments showed that Caveolin 2 regulates the activation of the small Rho GTPase CDC42 but apparently not RAC1 in human intestinal cells. CONCLUSIONS/SIGNIFICANCE: Our study outlines for the first time important regulation pathways in intestinal Salmonella infection pointing out that focal adhesion and organisation of actin cytoskeleton are regulated by microRNAs. Functional relevance is shown by miR-29a mediated Caveolin 2 regulation, modulating the activation state of CDC42. Further analysis of examined interactions may support the discovery of novel strategies impairing the uptake of intracellular pathogens.

Glycine-terminated dendritic amphiphiles for nonviral gene delivery.

Development of nonviral vectors for the successful application of gene therapy through siRNA/DNA transfection of cells is still a great challenge in current research. (1, 2) In the present study, we have developed multivalent polyglycerol dendron based amphiphiles with well-defined molecular structures that express controlled glycine arrays on their surfaces. The structure-activity relationships with respect to the siRNA complexation, toxicity, and transfection profiles were studied with synthesized amphiphilic polycations. Our findings revealed that a second-generation amphiphilic dendrimer (G2-octaamine, 4) that has eight amine groups on its surface and a hydrophobic C-18 alkyl chain at the core of the dendron, acts as an efficient vector to deliver siRNA and achieve potent gene silencing by investigating the knockdown of luciferase and GAPDH gene activity in HeLa cells. Interestingly, the amphiphilic vector is nontoxic even at higher ratio of N/P 100. To the best of our knowledge this is the first example of successful in vitro siRNA transfection using dendritic amphiphiles. We believe that this supramolecular complex may serve as a new promising alternative for nonviral siRNA delivery systems and will be investigated for in vivo siRNA delivery in the future.

Quantification and accurate normalisation of small RNAs through new custom RT-qPCR arrays demonstrates Salmonella-induced microRNAs in human monocytes.

BACKGROUND: Small interfering and non-coding RNAs regulate gene expression across all kingdoms of life. MicroRNAs constitute an important group of metazoan small RNAs regulating development but also disease. Accordingly, in functional genomics microRNA expression analysis sheds more and more light on the dynamic regulation of gene expression in various cellular processes. RESULTS: We have developed custom RT-qPCR arrays allowing for accurate quantification of 31 small RNAs in triplicate using a 96 well format. In parallel, we provide accurate normalisation of microRNA expression data based on the quantification of 5 reference snRNAs. We have successfully employed such arrays to study microRNA regulation during human monocyte differentiation as well as Salmonella infection. Besides well-known protagonists such as miR-146 or miR-155, we identified the up-regulation of miR-21, miR-222, miR-23b, miR-24, miR-27a as well as miR-29 upon monocyte differentiation or infection, respectively. CONCLUSIONS: The provided protocol for RT-qPCR arrays enables straight-forward microRNA expression analysis. It is fully automatable, compliant with the MIQE guidelines and can be completed in only 1 day. The application of these arrays revealed microRNAs that may mediate monocyte host defence mechanisms by regulating the TGF-ß signalling upon Salmonella infection. The introduced arrays are furthermore suited for customised quantification of any class of small non-coding RNAs as exemplified by snRNAs and thus provide a versatile tool for ubiquitous applications.

Deciphering the porcine intestinal microRNA transcriptome.

BACKGROUND: While more than 700 microRNAs (miRNAs) are known in human, a comparably low number has been identified in swine. Because of the close phylogenetic distance to humans, pigs serve as a suitable model for studying e.g. intestinal development or disease. Recent studies indicate that miRNAs are key regulators of intestinal development and their aberrant expression leads to intestinal malignancy. RESULTS: Here, we present the identification of hundreds of apparently novel miRNAs in the porcine intestine. MiRNAs were first identified by means of deep sequencing followed by miRNA precursor prediction using the miRDeep algorithm as well as searching for conserved miRNAs. Second, the porcine miRNAome along the entire intestine (duodenum, proximal and distal jejunum, ileum, ascending and transverse colon) was unraveled using customized miRNA microarrays based on the identified sequences as well as known porcine and human ones. In total, the expression of 332 intestinal miRNAs was discovered, of which 201 represented assumed novel porcine miRNAs. The identified hairpin forming precursors were in part organized in genomic clusters, and most of the precursors were located on chromosomes 3 and 1, respectively. Hierarchical clustering of the expression data revealed subsets of miRNAs that are specific to distinct parts of the intestine pointing to their impact on cellular signaling networks. CONCLUSIONS: In this study, we have applied a straight forward approach to decipher the porcine intestinal miRNAome for the first time in mammals using a piglet model. The high number of identified novel miRNAs in the porcine intestine points out their crucial role in intestinal function as shown by pathway analysis. On the other hand, the reported miRNAs may share orthologs in other mammals such as human still to be discovered.