Department of Surgery, Brigham and Women's Hospital, Boston, MA 02115, USA.
Exp Cell Res. 2010 Dec 10;316(20):3512-21. doi: 10.1016/j.yexcr.2010.07.007. Epub 2010 Jul 13.
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.
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.
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.
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.
肠道在功能和形态上表现出明显的昼夜节律，部分原因是肠细胞增殖的变化。这些节律背后的调节机制在很大程度上仍然未知。我们假设 microRNAs 参与调节这些节律，并专门研究了 microRNAs 在调节肠道增殖中的作用。
通过微阵列分析大鼠空肠中 microRNAs 的昼夜节律性，并通过 qPCR 进行验证。使用激光捕获显微切割和 qPCR 进一步定量分析肠道隐窝、绒毛和平滑肌中昼夜节律性 mir-16 的表达。通过组织学评估大鼠空肠的形态变化，通过溴脱氧尿苷免疫染色评估增殖。在稳定过表达 mir-16 的 IEC-6 细胞中，通过细胞计数和 MTS 测定评估增殖，通过流式细胞术评估细胞周期进程和细胞凋亡，通过 qPCR 和免疫印迹评估细胞周期基因表达。
mir-16 在光照后 6 小时（HALO 6）达到峰值，昼夜变化仅限于隐窝。隐窝深度和绒毛高度在与 mir-16 相反的 HALO 13-14 达到峰值。在 IEC-6 细胞中过表达 mir-16 抑制了特定的 G1/S 调节因子（细胞周期蛋白 D1-3、细胞周期蛋白 E1 和细胞周期蛋白依赖性激酶 6）并产生 G1 期阻滞。这些基因的蛋白表达在大鼠空肠中表现出昼夜节律性，与 mir-16 相反，在 HALO 11 到 17 之间达到峰值。
这是大鼠空肠特定 microRNAs 昼夜节律性的首次报道。我们的数据为抗增殖的 mir-16 与肠道增殖节律之间提供了联系，并指出 mir-16 是 jejunal 隐窝增殖的重要调节因子。这种功能对于将增殖和吸收能力与营养供应相匹配可能是必不可少的。
