Gastroenterology

Gastroenterology

Volume 139, Issue 5, November 2010, Pages 1654-1664.e1
Gastroenterology

Basic—Alimentary Tract
MicroRNAs Control Intestinal Epithelial Differentiation, Architecture, and Barrier Function

https://doi.org/10.1053/j.gastro.2010.07.040Get rights and content

Background & Aims

Whereas the importance of microRNA (miRNA) for the development of several tissues is well established, its role in the intestine is unknown. We aimed to quantify the complete miRNA expression profile of the mammalian intestinal mucosa and to determine the contribution of miRNAs to intestinal homeostasis using genetic means.

Methods

We determined the miRNA transcriptome of the mouse intestinal mucosa using ultrahigh throughput sequencing. Using high-throughput sequencing of RNA isolated by cross-linking immunoprecipitation (HITS-CLIP), we identified miRNA-messenger RNA target relationships in the jejunum. We employed gene ablation of the obligatory miRNA-processing enzyme Dicer1 to derive mice deficient for all miRNAs in intestinal epithelia.

Results

miRNA abundance varies dramatically in the intestinal mucosa, from 1 read per million to 250,000. Of the 453 miRNA families identified, mmu-miR-192 is the most highly expressed in both the small and large intestinal mucosa, and there is a 53% overlap in the top 15 expressed miRNAs between the 2 tissues. The intestinal epithelium of Dicer1loxP/loxP;Villin-Cre mutant mice is disorganized, with a decrease in goblet cells, a dramatic increase in apoptosis in crypts of both jejunum and colon, and accelerated jejunal cell migration. Furthermore, intestinal barrier function is impaired in Dicer1-deficient mice, resulting in intestinal inflammation with lymphocyte and neutrophil infiltration. Our list of miRNA-messenger RNA targeting relationships in the small intestinal mucosa provides insight into the molecular mechanisms behind the phenotype of Dicer1 mutant mice.

Conclusions

We have identified all intestinal miRNAs and shown using gene ablation of Dicer1 that miRNAs play a vital role in the differentiation and function of the intestinal epithelium.

Section snippets

Identification and Quantification of miRNAs

Jejunal mucosa was scraped from the longitudinally sliced intestine of CD1 mice (n = 4), and small RNAs were isolated using the mirVana miRNA kit (Ambion catalogue No. AM1560). Libraries were prepared using the Digital Gene Expression-Small RNA sample prep kit (Illumina, San Diego, CA, FC-102-1009) and sequenced on a Genome Analyzer II (Illumina). Trimmed reads were aligned to precursor miRNA sequence from miRBase (release 13.0), reference sequence (RefSeq) sequence (National Center for

Results

We quantified small RNAs from jejunal and colonic mucosa of adult wild-type mice using ultrahigh throughput sequencing. We aligned the resulting sequence reads to known miRNA precursor genes obtained from miRBase.16 Next, we verified that these sequence reads represented miRNAs and not degraded mRNAs by aligning them to the RefSeq database. As shown in Figure 1A, less than 20% of reads in the miRNA size range aligned to mRNAs, whereas more than 90% matched precursor miRNAs, indicating that our

Discussion

By combining the mRNA microarray data with HITS-CLIP-derived targeting information, we can identify a set of miRNA targets whose expression levels are affected in the small intestine by deletion of Dicer1, among them the aforementioned Cadherin 1 and Cathepsin B genes (see Supplementary Table 4 for summary). We analyzed transcription factors as potentially important targets because they control transcriptional networks and often play fundamental well-studied roles in processes such as

Acknowledgments

The authors thank Drs Merkenschlager, Gumucio, Rustgi, and Mourelatos for reagents; Elizabeth Helmbrecht and Karrie Brondell for maintenance of the mouse colony; Amber Horner for technical assistance; Dr Gary Swain, Jaclyn Twaddle, and the entire morphology core of the Penn Center for Molecular Studies in Digestive and Kidney Disease (DK-050306) for reagents and technical assistance; Dr Marie Hildebrandt, Markiyan Doliba, and Christopher Morgan for technical assistance; and Dr John Le Lay for

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    Conflicts of interest The authors disclose no conflicts.

    Funding Supported by T32-HD007516 (to L.B.M.) and NIDDK grant R01-DK053839 (to K.H.K.).

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