Ascl2转录调节CDX2表达促肠上皮细胞向杯状细胞表型分化的分子机制研究

Achaete scute-like 2 (Ascl2), a basic helix-loop-helix (bHLH) transcription factor, is a downstream target of Wnt signaling that controls the fate of intestinal cryptic stem cells. Our previous study indicated that Ascl2 knockdown in the intestinal epithelial cells led to the significant increase of MUC2 expression in both levels of protein and mRNA of which is a typical marker of goblet cells, and CDX2 expression in protein level of which is a typical gene of intestinal epithelial differentiation. The molecular mechanism that Ascl2 modulates their expression is unclear. The study is to investigate how Ascl2 transcriptionally regulates CDX2 expression in the intestinal epithelial cells and further activates MUC2 expression using following experiments: 1.To identify the important and pivotal DNA sequence residing at the CDX2 proximal promoter through the serial truncated CDX2 proximal promoter and luciferase reporter assay; 2.To analyse the potential the cis-elements at the CDX2 proximal promoter of some important transcription factor, especially Ascl2, using the commercially available nuclear transcription factors analysis database(s); 3. To further mutate the potential Ascl2 binding cis-element(s) at the CDX2 proximal promoter and compare the difference of transcriptional activity between the CDX2 proximal promoter with wild-type Ascl2 cis-element(s) and the CDX2 proximal promoter with mutated Ascl2 cis-element(s); 4. To confirm the binding between Ascl2 and the CDX2 proximal promoter using EMSA experiment in vivo and ChIP array in vivo; 5. To provide the evidence of correlation between Ascl2 and CDX2, and between Ascl2 and MUC2 expression in the clinical samples of colorectal carcinoma and gives the indirect messages to support the idea which Ascl2 represses CDX2 expression and further inhibits MUC2 expression in the intestinal epithelial cells. The study is not only helpful to understand the transcriptional mechanism of Ascl2 modulating CDX2 expression, but will provide the novel knowledge of the relationship between the maintenance of intestinal cryptic stem cells and their differentiation.

Ascl2作为Wnt信号的靶基因，是控制肠粘膜隐窝干细胞命运的转录因子。前期研究发现抑制结肠上皮细胞Ascl2的表达导致肠杯状细胞标志MUC2和肠分化基因CDX2的表达增加，但其调控分子机制不清。本研究拟通过启动子缺失片段报告基因系统初步分析Ascl2对CDX2的转录调控的核心序列；应用核转录因子数据库筛选出可能发挥作用的顺式作用元件及转录因子；进一步定点突变可能的顺式作用元件，比较Ascl2对突变体与野生型的CDX2启动子活性的差异；再分别用EMSA实验、ChIP实验在体外、体内水平验证Ascl2与CDX2启动子区的顺式作用元件的存在结合调节作用；并在结肠癌组织标本中研究Ascl2、CDX2 及肠杯状细胞标志MUC2等的表达水平之间的相关性，以获得Ascl2调控CDX2表达的组织学水平的依据。本课题研究通过揭示Ascl2转录调控CDX2表达的分子机制，提供干性维持与细胞分化之间的新认识。

Ascl2作为Wnt信号的靶基因，是控制肠粘膜隐窝干细胞命运的转录因子。研究发现抑制结肠上皮细胞Ascl2的表达导致肠杯状细胞标志MUC2和肠分化基因CDX2的表达增加，后期进一步验证结肠癌细胞中Ascl2调控CDX2的分子机制，即Ascl2通过结合E-box元件转录抑制CDX2，从而抑制肠道上皮细胞向杯状细胞的分化。在已知Ascl2是调节长上皮细胞向杯状细胞分化的关键因子前提下，进一步对Ascl2上游调控进行研究。通过ChIP实验和双荧光素酶报告系统证明HIF-1α诱导Ascl2转录激活，并鉴定HIF-1α与.Ascl2的结合位点，以及后期验证YAP1促进除KLF5之外的相关干性因子的表达可能通过诱导Ascl2实现。本课题研究通过揭示Ascl2转录调控CDX2表达的分子机制以及Ascl2上游调控机制，提供干性维持与细胞分化之间的新认识。