Critical Role of Mybl2 in Spontaneous and Short-Chain Fatty Acid-Induced Intestin

描述（来自申请人）：结肠上皮细胞分化是由干细胞和/或祖细胞的重编程驱动的，因为它们沿着隐窝轴沿着向上迁移。这一过程在来源于人结肠腺癌的永生化细胞系中重现，该细胞系在培养中以不成熟状态增殖，但可以由短链脂肪酸（SCFA）丁酸酯诱导（膳食纤维在人体结肠中发酵的产物），或接触抑制生长分化为吸收性（Caco-2），杯状（HT 29 Cl 16 E）或分泌（HT 29 Cl 19 A）结肠上皮细胞。我们的研究小组通过表征这些细胞谱系在分化时的基因表达谱，为驱动这种成熟的机制提供了关键的见解。为了确定基因的表达改变是根本的肠上皮细胞的重编程，因为他们经历 由于丁酸盐等生理调节剂诱导的成熟，我们将重点放在 体内结肠上皮细胞生长停滞和谱系特异性分化期间表达持续下调。在增殖的结肠上皮细胞中通过siRNA抑制一种这样的基因Mybl 2（已被认为调节干细胞和其他细胞类型中的关键增殖和分化决定的分子）诱导分化特异性基因的子集的表达。该提案的目的集中于确定Mybl 2是否通过对增殖和分化途径施加平行控制来调节结肠上皮细胞重编程，包括由SCFA丁酸盐诱导的重编程，并阐明Mybl 2本身在结肠细胞成熟期间是如何调节的。具体目标1和2将确定Mybl 2抑制是否可以通过增殖和分化特异性基因的协调调节来促进或其过表达是否可以阻止结肠细胞成熟。具体目标#1追求我们鉴定参与促进或阻止结肠细胞成熟的基因子集，其在Mybl 2敲低或过表达时改变，具体目标#2将揭示哪些基因启动子在成熟的结肠细胞中被Mybl 2结合和调节。虽然转录激活和抑制占许多分化特异性基因表达的变化，转录后的调控microRNA（miRNA）提供了另一个关键水平的基因调节在结肠细胞分化。我们发现Mybl 2启动子在分化的结肠上皮细胞中没有显著下调，因此假设miRNA可能在成熟过程中调节其表达。我们已经利用新的生物信息学技术，以确定在Mybl 2 3'非翻译区（UTR）的潜在的miRNA的目标。这些潜在目标：1）通过计算机分析预测结合一种或多种已知的miRNA和2）形成显著的RNA二级结构，如预测的自由能< 0所示。因此，这些候选miRNA靶区域具有结合调节所有三种结肠细胞谱系中分化的基本机制的miRNA的潜力。这种以靶标为中心的方法将用于鉴定在结肠细胞分化期间调节Mybl 2表达的特定miRNA及其靶标。这种方法，而不是筛选已知的miRNA的表达，将分离和鉴定与结肠细胞分化具有生物学相关性的已知和未鉴定的miRNA。因此，目的3将首先通过测定Mybl 2 3' UTR中的预测的miRNA靶在插入分化的结肠上皮细胞中的荧光素酶的3' UTR中时抑制荧光素酶表达的能力来确定它们是否是功能性的，如已经由一种这样的预测的miRNA靶的初步数据所指示的。然后将功能靶标固定在链霉亲和素/琼脂糖上，以从分化的结肠上皮细胞的小RNA中物理捕获miRNA（已知的或新的）。因此，拟议的研究将揭示几种结肠上皮细胞谱系用来调节增殖或分化决定的基本机制。

DESCRIPTION (from the applicant): Colonic epithelial cell differentiation is driven by reprogramming of stem and/or progenitor cells as they migrate upward along the crypt axis. This process is recapitulated in immortalized cell lines derived from human colon adenocarcinomas that proliferate in an immature state in culture yet can be induced by the short chain fatty acid (SCFA) butyrate (a product of dietary fiber fermentation in the human colon), or contact inhibition of growth to differentiate into absorptive (Caco-2), goblet (HT29 Cl16E), or secretory (HT29 Cl19A) colonic epithelial cells. Our group has provided key insights into mechanisms that drive this maturation by characterizing the gene expression profiles of these cell lineages as they differentiate. To identify genes for which altered expression is fundamental to the reprogramming of intestinal epithelial cells as they undergo maturation induced by physiological regulators such as butyrate, we focused on sequences that are consistently downregulated in expression during growth arrest and lineage specific differentiation of colonic epithelial cells in vivo. Suppression of one such gene, Mybl2, a molecule that has been suggested to regulate critical proliferation and differentiation decisions in stem cells and other cell types, by siRNA in proliferating colon epithelial cells induces expression of a subset of differentiation-specific genes. The aims of this proposal focus on determining whether Mybl2 modulates colon epithelial cell reprogramming, including that induced by the SCFA butyrate, by exerting parallel control over proliferation and differentiation pathways and elucidating how Mybl2 is itself regulated during colon cell maturation. Specific aims 1 and 2 will determine whether Mybl2 suppression can promote, or its overexpression can prevent, colon cell maturation through the coordinate regulation of proliferation and differentiation-specific genes. Specific aim #1 pursues our identification of a subset of genes, involved in promoting or preventing colon cell maturation, that is altered upon Mybl2 knockdown or overexpression, and specific aim #2 will reveal which gene promoters are bound and regulated by Mybl2 in maturing colon cells. Although transcriptional activation and repression account for many differentiation-specific gene expression changes, post-transcriptional regulation by microRNAs (miRNAs) provides another critical level of gene modulation during colon cell differentiation. We have found that the Mybl2 promoter is not significantly downregulated in differentiating colon epithelial cells and have therefore hypothesized that miRNAs may regulate its expression during maturation. We have utilized novel bioinformatic techniques to identify potential miRNA targets in the Mybl2 3' untranslated region (UTR). These potential targets: 1) are predicted by computer analysis to bind one or more known miRNAs and 2) form significant RNA secondary structure as indicated by a predicted free energy < 0. These candidate miRNA target regions therefore have the potential to bind miRNA's that regulate fundamental mechanisms of differentiation in all three colon cell lineages. This target-centered approach will be used to identify specific miRNAs and their targets that regulate Mybl2 expression during colon cell differentiation. This approach, rather than a screening of expression of known miRNAs, will isolate and identify known and as well as unidentified miRNAs of biological relevance to colonic cell differentiation. Therefore, aim 3 will first determine whether predicted miRNA targets in the Mybl2 3' UTR are functional by assaying their ability to suppress luciferase expression when inserted into the 3' UTR of luciferase in differentiating colonic epithelial cells, as already indicated by preliminary data for one such predicted miRNA target. Functional targets will then be immobilized on streptavidin/agarose to physically capture miRNAs (known or novel) from small RNAs of differentiating colonic epithelial cells. The proposed studies will therefore reveal fundamental mechanisms utilized by several colon epithelial cell lineages to regulate the decision to proliferate or differentiate.