Abstract
MicroRNAs play extensive roles in cellular development. Analysis of the microRNA expression pattern during intestinal cell proliferation in early life is likely to unravel molecular mechanisms behind intestinal development and have implications for therapeutic intervention. In this study, we isolated mouse intestinal crypt cells, examined the differences in microRNA expression upon IGF-1 stimulated proliferation and identified miR-103 as a one of the key regulators. Mouse intestinal crypt cells were cultured and treated with IGF-1 for 24 h. MicroRNA microarray showed that multiple microRNAs are regulated by IGF-1, and miR-103 was the most sharply down-regulated. Expression of miR-103 in mouse intestinal crypt cells was confirmed by real-time Q-PCR. Sequence analyses showed that, among the 1040 predicted miR-103 target genes, CCNE1, CDK2, and CREB1 contain complementary sequences to the miR-103 seed region that are conserved between human and mouse. We further demonstrated that miR-103 controls the expression level of these three genes in mouse crypt cells by luciferase assay and immunoblotting assay. Taken together, our data suggest that in mouse intestinal crypt cells, miR-103 is part of the G1/S transition regulatory network, which targets CCNE1, CDK2, and CREB1 during IGF-1 stimulated proliferation.
Highlights
The small intestinal epithelium is a major site for nutrient absorption and serves as an important barrier to prevent exogenous pathogens from entering the body
Mouse intestinal crypt cell culture This study complied with the Guide for the Use and Care of Laboratory Animals and the protocol was approved by the Institutional Animal Care and Use Committee at University of California, Davis, which is accredited by the American Association for the Accreditation of Laboratory Animal Care (AAALAC)
IGF-1 is a 7.5 kD polypeptide consisting of 70 amino acids, and found to be present in human milk [25]
Summary
The small intestinal epithelium is a major site for nutrient absorption and serves as an important barrier to prevent exogenous pathogens from entering the body. The small intestine is a highly dynamic and well-structured tissue which compartmentalizes into villi and the crypts of Lieberkuhn (crypts). The intestine regenerates itself throughout the life as the intestinal epithelial cells regularly shed off from the villi. This continuous cell renewal process is achieved by pluripotent epithelial stem cells which populate the specialized proliferative units known as the crypts. The crypts are localized at the intervillus region, and formed as the result of epithelial invaginations towards the basolateral side of the epithelium. Rodent studies have revealed that crypt structures form during the first few days after birth, and continue to develop during the several weeks. Cummins et al [1] found that crypt fission ( called branching, which is a process of physiologic mechanism of crypt reproduction) is present predominantly during infancy, but not during later developmental stages, further supporting the significance of infancy for crypt development
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