MicroRNA mir-16 is anti-proliferative in enterocytes and exhibits diurnal rhythmicity in intestinal crypts.
OBJECTIVE
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.
Balakrishnan
Anita
A
Department of Surgery, Brigham and Women's Hospital, Boston, MA 02115, USA. anita.balakrishnan@doctors.org.uk
Stearns
Adam T
AT
Park
Peter J
PJ
Dreyfuss
Jonathan M
JM
Ashley
Stanley W
SW
Rhoads
David B
DB
Tavakkolizadeh
Ali
A
D000818:Animals; D049108:Cell Enlargement; D002460:Cell Line; D049109:Cell Proliferation; D002940:Circadian Rhythm / Q000235:genetics*; D051361:Cyclin-Dependent Kinase 6 / Q000235:genetics / Q000378:metabolism; D016213:Cyclins / Q000235:genetics / Q000378:metabolism; D004247:DNA / Q000096:biosynthesis; D020895:Enterocytes / Q000166:cytology* / Q000378:metabolism*; D016193:G1 Phase / Q000235:genetics; D015870:Gene Expression / Q000235:genetics; D007413:Intestinal Mucosa / Q000033:anatomy & histology / Q000166:cytology / Q000378:metabolism; D007583:Jejunum / Q000033:anatomy & histology / Q000166:cytology / Q000378:metabolism*; D008297:Male; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D009130:Muscle, Smooth / Q000378:metabolism; D017440:Photoperiod; D051381:Rats; D017207:Rats, Sprague-Dawley; D014162:Transfection
D016213:Cyclins; C547573:MIRN16 microRNA, rat; D035683:MicroRNAs; D004247:DNA; C495913:Cdk6 protein, rat; D051361:Cyclin-Dependent Kinase 6