Transcriptional Regulation Of CDX2 Gene By Ascl2 In Human Colon Cancer Epithelial Cells

Background and ObjectiveIn recent years, the morbidity and mortality of malignant tumor patients keep on rising in China. As the common type in gastrointestinal tract, the incidence and mortality of colon cancer rank in the top ten among all malignant tumors. The occurrence of colon cancer has differences within gender, regional and ethnic. The colon cancer’s incidence in men is higher than that in women. In North American and Western Europe, the incidence of colon cancer is higher than that in African and Asian. In African-Americans, the incidence of this disease is higher than that in Caucasians in the same distraction. Achaete scute-like 2(Ascl2), a downstream target of WNT signaling that controls the fate of intestinal cryptic stem cells and colorectal cancer(CRC) progenitor cells. Our preliminary study finding, Ascl2 loss-of-function in CRC cells promoted the differentiation of CRC cells into a globlet cell phenotype determined by the up-regulated expression of globlet cell-cpecific markers MUC2, TFF3 and CDX2. Caudal-related homemaking transcription factor 2 that can be expressed specificity in gastrointestinal epithelial cells is a newly discovered nuclear transcription factor. CDX2 is involved in controlling embryonic cell differentiation and development and maintaining the stability of the adult intestinal cells. The expression of CDX2 is closely related to the occurrence of gastrointestinal tumor, its abnormal expression in gastric and esophageal mucous epithelial cells can induce the occurrence of intestinal metaplasia. At the same time, CDX2 can make the migration and proliferation of tumor cells slowly,it can be used as a tumor supressing factor of intestinal tumor inhbiting its occurence.However, the mechanism of CDX2 gene inhibiting intestinal tumorigenesis is not yet clear. Through NCBI database and cold spring harbor database analysis, we found there have abundent E-box elements. Our premilinary study confirmed, suppressed expression of Ascl2 can lead to appear globlet cell phenotypic of colon epithelial cells. Ascl1 as the homologous genes of Ascl2 can specific combine E-box element. We hypothesized that Ascl2 may also trnscriptional regulate the expression of CDX2 gene through the mechanism. To discuss the transcriptional regulation machanism of CDX2 by Ascl2 in human colon cancer epithelial cells, the Ascl2 knockdowned CRC cells were transfected with CDX2 promoter constructs and used for luciferase assays and chromatin immunoprecipitation(ChIP) assays.Methods1. The region of CDX2 proximal promoter(-2000~1) were amplified by PCR using genomic DNA of colon cancer cell line LS174 T as template.2. The potential binding sites of CDX2 promoter region were predicted and analysed by TF research 1.3 and cold spring harbor databases.3. According to the position of E-box components in CDX2 promoter region, we determined the amplification area of human CDX2 promoter deletion fragments. DNA fragments were amplified by PCR using DNA of CDX2 proximal promoter(-2000~1) area as template.4. pGL3-Basic plasmid was used to construct the luciferase report gene plasmids of CDX2 promoter region and corresponding delation fragments and confirmed by sequencing.5. The relative luciferase activity were detected after transfection of recominant plasmids into shRNA-Ascl2/LS174 T and shRNA-Ascl2/LS174 T cells.6. The combination of transcriptional factor Ascl2 and CDX2 promoter were detected by chromatin immune coprecipitation assay.Results1. CDX2 proximal promoter(-2000~1) area were successfully amplified by PCR.2. There have eight E-box components and several conservation sequences of potential transcription factor binding sites analysed by TF Search 1.3 and cold spring harbor databases.3. The delation fragments of CDX2 promoter were successfully amplified by PCR.4. Eight luciferase reporter gene plasmids were successfully constructed and the insert fragment sequences were proved to be corrected by enzyme digestion and sequencing.5. The relative luciferase activity of constructed plasmids pGL3-CDX2-C1~C8 in shRNA-Ascl2/LS174 T and shRNA-Ctr/LS174 T cells were detected by luciferase reporter gene detection system. The constructed plasmids had lower luciferase activity in shRNA- Ctr/LS174 T cells than in shRNA-Ascl2/LS174 T cells(P<0.01). As the fragments to be shortened, the luciferase activity is higher and higher.6. CDX2 proximal promoter may had several Ascl2 binding fragments confirmed by chromatin immune copecipitation experiment. The DNA fragments within(-841~-646、-291~-134) can be bind by Ascl2 and showed significant reduction in shRNA-Ascl2/ LS174 T cells than in shRNA-Ascl2/LS174 T cells.Conclusions1. The luciferase reporter gene expressing vector pGL3-CDX2-C1~C8 were successfully constructed.2. Ascl2 may transriotional suppresses the expression of CDX2 gene by directly combining to its proximal promoter in human colon cancer epithelial cells.