Ascl2转录调控人结肠癌上皮细胞内CDX2基因表达的研究

Transcriptional regulation of CDX2 gene expression by Ascl2 in colon cancer epithelial cells

目的探讨Ascl2对人结肠癌上皮细胞中CDX2基因的转录调控作用。方法预测CDX2启动子的转录因子结合位点,构建包含8个不同长度的CDX2近端启动子的荧光素酶报告基因质粒,分别命名为pGL3-CDX2-C1-8;将各质粒分别转染到shRNA-Ascl2/LS174T和shRNA-Ctr/LS174T细胞中,检测细胞中的双荧光素酶活性。采用染色质免疫共沉淀(ChIP)技术,用Ascl2抗体免疫沉淀与CDX2启动子区DNA片段相结合的Ascl2,PCR扩增CDX2启动子的特异性序列。结果转录因子数据库预测到CDX2近端启动子区含多个E-box结合位点和潜在的Nkx-2、GATA-1、CdxA、Sox-5、SRY等顺式作用元件。双酶切及测序结果证实pGL3-CDX2-C1-8重组质粒插入片段序列均正确。重组质粒在shRNAAscl2/LS174T细胞中的荧光素酶活性明显高于shRNA-Ctr/LS174T细胞(P<0.01)。随着CDX2启动子片段的缩短,荧光素酶活性呈升高趋势。ChIP实验证实在CDX2近端启动子存在与Ascl2相结合的片段,在shRNA-Ascl2/LS174T细胞中,Ascl2与CDX2启动子区域中-841^-646、-291^-134、-7^+138片段结合的DNA片段明显少于对照shRNA-Ctr/LS174T细胞。结论 Ascl2在结肠癌上皮细胞中可能通过与CDX2近端启动子的直接结合而达到转录阻遏CDX2基因表达的目的。

Objective To investigate the transcriptional regulation of caudal-related homeodomain transcription factor 2（ CDX2） gene expression by Achaete scute-like 2（ Ascl2） in colon cancer epithelial cells. Methods Transcription factor binding sites of CDX2 promoter were predicted. Vector pGL3-Basic was used to construct 8 recombinant plasmids（ pGL3-CDX2-C1 to pGL3-CDX2-C8） containing different truncated CDX2 proximal promoters. After transfection of recombinant plasmids into shRNA-Ascl2 /LS174 T and shRNACtr /LS174 T cells,the activity of those luciferase reporters was detected by luciferase reporter assay. We used chromatin immunoprecipitation（ ChIP） assay with an anti-Ascl2 antibody to immunoprecipitate the possible Ascl2-binding DNA fragments within CDX2 proximal promoter. The immunoprecipitates were amplified through PCR using specific CDX2 primers. Results Predicted by transcription factor databases,the CDX2 proximal promoter contained the abundant E-box（ s） and the probable cis-regulatory elements for Nkx-2,GATA-1,CdxA,Sox-5 and SRY. The inserts within those luciferase reporters were proved to be correct by enzyme digestion and sequencing. These luciferase reporters had higher luciferase activity in shRNA-Ascl2 /LS174 T cells than in shRNA-Ctr /LS174 T cells（P 0. 01）. However,further truncation of the CDX2 promoter resulted in an increased luciferase activity. The specific DNA fragments in CDX2 proximal promoter were detected in the immunoprecipitates by the anti-Ascl2 antibody. The semi-quantitation of specific DNA fragments within- 841to-646,-291 to-134 or-7 to ＋138 of CDX2 proximal promoter showed their significant reduction in shRNAAscl2 /LS174 T cells as compared to shRNA-Ctr /LS174 T cells（P 0. 05）. Conclusion Ascl2 transcriptionally suppresses CDX2 gene expression by direct binding to its proximal promoter in colon cancer epithelial cells.