Elsevier

Experimental Cell Research

Volume 316, Issue 20, 10 December 2010, Pages 3512-3521
Experimental Cell Research

Research Article
MicroRNA mir-16 is anti-proliferative in enterocytes and exhibits diurnal rhythmicity in intestinal crypts

https://doi.org/10.1016/j.yexcr.2010.07.007Get rights and content

Abstract

Background and aims

The intestine exhibits profound diurnal rhythms in function and morphology, in part due to changes in enterocyte proliferation. The regulatory mechanisms behind these rhythms remain largely unknown. We hypothesized that microRNAs are involved in mediating these rhythms, and studied the role of microRNAs specifically in modulating intestinal proliferation.

Methods

Diurnal rhythmicity of microRNAs in rat jejunum was analyzed by microarrays and validated by qPCR. Temporal expression of diurnally rhythmic mir-16 was further quantified in intestinal crypts, villi, and smooth muscle using laser capture microdissection and qPCR. Morphological changes in rat jejunum were assessed by histology and proliferation by immunostaining for bromodeoxyuridine. In IEC-6 cells stably overexpressing mir-16, proliferation was assessed by cell counting and MTS assay, cell cycle progression and apoptosis by flow cytometry, and cell cycle gene expression by qPCR and immunoblotting.

Results

mir-16 peaked 6 hours after light onset (HALO 6) with diurnal changes restricted to crypts. Crypt depth and villus height peaked at HALO 13–14 in antiphase to mir-16. Overexpression of mir-16 in IEC-6 cells suppressed specific G1/S regulators (cyclins D1–3, cyclin E1 and cyclin-dependent kinase 6) and produced G1 arrest. Protein expression of these genes exhibited diurnal rhythmicity in rat jejunum, peaking between HALO 11 and 17 in antiphase to mir-16.

Conclusions

This is the first report of circadian rhythmicity of specific microRNAs in rat jejunum. Our data provide a link between anti-proliferative mir-16 and the intestinal proliferation rhythm and point to mir-16 as an important regulator of proliferation in jejunal crypts. This function may be essential to match proliferation and absorptive capacity with nutrient availability.

Introduction

Circadian rhythms (24-h oscillations) play a key role in the regulation of numerous physiological functions. Circadian rhythmicity of up to 10% of gene transcripts and an even greater fraction of proteins indicate the involvement of both transcriptional and translational pathways [1], [2], [3], [4], [5]. Regulation at both the transcriptional and post-transcriptional levels suggests a role for microRNAs in this process. MicroRNAs are non-coding RNAs able to silence numerous genes simultaneously. Bioinformatics analysis suggests that up to 30% of mammalian gene transcripts are regulated by microRNAs, short non-coding RNAs [6], [7], [8], [9]. microRNAs suppress protein expression following recognition of complementary sequences on the 3′UTR (untranslated region) of target genes, either by inducing mRNA cleavage (which manifests as changes in mRNA levels) or inhibiting translation (manifesting as changes in protein levels) [10], [11], [12]. The presence of the target sequence for each microRNA on multiple genes permits simultaneous regulation of protein expression from numerous genes by a single microRNA [6], [13], [14]. The postulated role of microRNAs in “fine-tuning” gene expression suggests that they also contribute to coordinating the circadian rhythmicity of many genes and proteins [15], [16], [17], [18].

The intestine displays profound rhythmicity of morphology, resulting in peak absorptive function (e.g. for glucose) coinciding with maximal nutrient delivery to the bowel [19], [20]. The number of enterocytes per villus also exhibits a diurnal rhythmicity, with an increase about the time of maximal nutrient availability [21]. Similar rhythmicity has been reported in human gastrointestinal mucosa [22], [23]. The exact pathways coordinating rhythmicity in proliferation are presently unknown.

We hypothesize that microRNAs are integral components for mediating circadian rhythms in intestinal proliferation, morphology, and function. To investigate this, we profiled microRNAs in the intestine of ad libitum fed rats using oligonucleotide arrays. The anti-proliferative microRNA mir-16 was expressed in both crypt and villus enterocytes but exhibited circadian rhythmicity only in the crypts. The cell cycle regulators Ccnd1, Ccnd2, Ccnd3, Ccne1, and Cdk6 also exhibited circadian rhythmicity but in antiphase to mir-16. An anti-proliferative role for mir-16 was supported by its ability to inhibit proliferation and decrease expression of genes involved in cell cycle regulation when overexpressed in rat IEC-6 cells. These studies point to mir-16 as a potentially important microRNA in regulating circadian rhythms in the intestine.

Section snippets

Animal studies

All animal study protocols were prospectively approved by the Harvard Medical Area Standing Committee on Animals.

Sprague–Dawley rats (50 males, 7 weeks old) were purchased from Harlan World (Indianapolis, IN) and acclimatized to a 12:12-h light: dark photoperiod for 5 days with ad libitum access to food and water. Time is designated as hours after light onset (HALO), with HALO 0 at 7 am (lights on). Rats were injected with BrdU (5-bromo-2-deoxyuridine, 50 mg/kg; Sigma, St Louis, MO) 1 h before

microRNAs exhibit diurnal rhythmicity in rat intestine

Of 238 microRNAs tested on in situ hybridization arrays, 13 microRNAs exhibited ≥ 2-fold difference between peak and trough values (range 2.0- to 3.4-fold; q < 0.05), 8 of which are conserved among human, mouse and rat and were therefore selected for further evaluation. Real-time PCR (qPCR) confirmed circadian rhythmicity for mir-16, mir-20a and mir-141 as determined by the cosinor procedure, with a 24-hour periodicity. Peak expression of these three microRNAs occurred between HALO 4 and 6,

Discussion

This study is the first to profile microRNA expression in rat jejunum as well as to establish rhythmic expression of specific microRNAs. In particular, our data supports a role for the anti-proliferative microRNA mir-16 in the intestinal proliferation rhythm. In support of this, we have shown that mir-16 expression peaks at HALO 6, coincident with the troughs in villus height and in crypt depth and cell number. mir-16 rhythmicity was also restricted to intestinal crypts, the primary site of

Acknowledgments

The authors gratefully acknowledge the excellent technical assistance of Jan Rounds and Roger Lis in the experimental procedures.

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