Research ArticleMicroRNA mir-16 is anti-proliferative in enterocytes and exhibits diurnal rhythmicity in intestinal crypts
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.
References (63)
- et al.
Microarray analysis and organization of circadian gene expression in Drosophila
Cell
(2001) - et al.
Circadian cycling of the mouse liver transcriptome, as revealed by cDNA microarray, is driven by the suprachiasmatic nucleus
Curr. Biol.
(2002) - et al.
Circadian regulation of gene expression systems in the Drosophila head
Neuron
(2001) - et al.
Coordinated transcription of key pathways in the mouse by the circadian clock
Cell
(2002) - et al.
Circadian orchestration of the hepatic proteome
Curr. Biol.
(2006) - et al.
Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets
Cell
(2005) - et al.
The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14
Cell
(1993) - et al.
Posttranscriptional regulation of the heterochronic gene lin-14 by lin-4 mediates temporal pattern formation in C. elegans
Cell
(1993) - et al.
Animal microRNAs confer robustness to gene expression and have a significant impact on 3′UTR evolution
Cell
(2005) - et al.
microRNA modulation of circadian-clock period and entrainment
Neuron
(2007)