Further analysis in independent Caucasian populations and functional analysis are needed to answer this question. Host animals represent habitats for the diverse microbial ecosystems. The gastrointestinal tract, which harbors an abundant microbial population, is the most heavily colonized organ. Insights into the composition of microbial communities, microbe-host molecular interactions, and the impact of microbiota on developmental/functional features of the host have been acquired from studies on germ-free animals using genomic and associated computational methods. MicroRNAs, discovered in 1993, are small non- coding RNAs that post-transcriptionally regulate gene expression by binding to the 39-untranslated regions of target mRNAs. Such binding is not homologous, allowing a single miRNA to potentially regulate hundreds of genes. Increasing evidence has raised miRNAs as an important regulator of many cellular functions, yet any role for miRNAs in microbiota-host interactions remains conjectural. To address this topic, we investigated whether miRNAs are INCB18424implicated in the gut microbiota-mediated regulation of host gene expression. To determine if microbiota modulate expression of miRNAs in the host, germ-free mice were colonized with the microbiota from pathogen-free mice as previously described. Comparative profiling of miRNA expression using miRNA arrays revealed significantly different signal intensities for 1 and 10 probe sets, representing one and eight miRNAs that were differently expressed in the ileum and the colon, respectively, INCB28060 of colonized mice compared to germ-free littermates. To increase the stringency of the prediction, the Matchminer program was used to identify target genes that were predicted by at least two algorithms. This bioinformatic approach revealed 164 potentially downregulat- ed target genes for the upregulated mmu-miR-298 in the ileum. These potential miRNA target genes predicted by at least two algorithms will be compared with the genes that were dysregulated during colonization. To identify host genes dysregulated during microbial coloniza- tion, a DNA microarray was performed, exploring significantly different signal intensities for 124 and 302 probe sets, representing 97 and 241 dysregulated genes in the ileum and the colon, respectively. It is worth to note that data obtained from miRNA arrays and DNA microarray revealed higher numbers of miRNAs and genes differentially expressed in the colon than that observed in the ileum, possibly reflecting bacterial load, which increases gradually from the stomach toward the small intestine to attain maximum in the colon. Crossing the DNA microarray-detected dysregulated genes with the potential targets of dysregulated miRNAs predicted by at least two algorithms as described above revealed a single upregulated gene, Abcc3, potentially targeted by mmu-miR-665, for the colon, whereas no overlapping gene was found for the ileum. The up- regulation of Abcc3 during colonization was then validated by qRT- PCR and Western blotting. Figure 2 shows that colonization of mice with microbiota significantly increased expression of Abcc3 in the colon at both mRNA and protein levels. Together, by coupling a DNA array with a microRNA array accompanied with the computational approach, we identified Abcc3 as a dysregulated miRNA target gene during colonization. To directly examine the regulation of Abcc3 by mmu-miR-665, RAW 264.7 cells were transfected with vehicle, or a mmu-miR- 665 precursor, or a control miRNA precursor. As shown in Figure 3A and B, mmu-miR-665 significantly inhibited Abcc3 expression at both mRNA and protein levels as assessed by qRT- PCR and Western blotting, respectively.