CAJ | 학술논문

目的探讨结缔组织生长因子(CTGF)是否为T细胞因子4(TCF4)的靶基因,参与调控结直肠癌细胞Wnt/β-连环蛋白(β-catenin)通路.方法构建CTGF启动子质粒pGL3-CTGF,联合TCF4显性负性突变体表达质粒pcDNA3-dnTCF4,转染结肠癌细胞株SW480,双荧光素酶实验分析CTGF启动子转录活性,染色质免疫共沉淀(ChIP)法验证CTGF启动子区与TCF4的相互作用.结果 SW480转染pGL3-CTGF质粒后荧光素酶活性(0.78±0.04)显著高于对照组转染空白质粒(0.01±0.00)(P＜0.01)；同时转染阻断β-catenin/TCF4信号通路的pcDNA3-dnTCF4质粒和pGL3-CTGF质粒,SW480细胞的荧光素酶活性(0.26±0.03)比单纯转染pGL3-CTGF质粒显著下降(P＜0.05).ChIP-聚合酶链反应(PCR)证实CTGF为TCF4的直接作用靶基因.结论 CTGF是β-catenin-TCF4信号通路的下游靶基因,TCF4直接结合于CTGF启动子区.
목적 탐토결체조직생장인자(CTGF)시부위T세포인자4(TCF4)적파기인,삼여조공결직장암세포Wnt/β-련배단백(β-catenin)통로.방법 구건CTGF계동자질립pGL3-CTGF,연합TCF4현성부성돌변체표체질립pcDNA3-dnTCF4,전염결장암세포주SW480,쌍형광소매실험분석CTGF계동자전록활성,염색질면역공침정(ChIP)법험증CTGF계동자구여TCF4적상호작용.결과 SW480전염pGL3-CTGF질립후형광소매활성(0.78±0.04)현저고우대조조전염공백질립(0.01±0.00)(P＜0.01)；동시전염조단β-catenin/TCF4신호통로적pcDNA3-dnTCF4질립화pGL3-CTGF질립,SW480세포적형광소매활성(0.26±0.03)비단순전염pGL3-CTGF질립현저하강(P＜0.05).ChIP-취합매련반응(PCR)증실CTGF위TCF4적직접작용파기인.결론 CTGF시β-catenin-TCF4신호통로적하유파기인,TCF4직접결합우CTGF계동자구.
Objective To investigate whether connective tissue growth factor (CTGF),which is involved in the regulation of Wnt/β-catenin signaling pathway,is the target gene of T cell factor-4 (TCF4) in colorectal cancer (CRC) cells.Methods The promoter vector of CTGF (pGL3-CTGF) was constructed and transfected into CRC cell lines SW480,together with or without dominant negative TCF4 vector (pcDNA3-dnTCF4).Then the promoter activity of CTGF was detected using dual luciferase reporter assays.Chromatin immunoprecipitation (ChIP) was used to determine the interaction between TCF4 and CTGF promoter in SW480 cells.Results The luciferase activities of SW480 cells transfected with pGL3-CTGF were significantly higher than the blank control (0.78 ± 0.04 versus 0.01 ± 0,P ＜ 0.01).Cells co-transfected with pcDNA3-dnTCF4 could lead to significant suppression of the luciferase activity,comparing to the cells transfected with pGL3-CTGF alone (0.26 ±0.03 versus 0.78 ±0.04,P ＜0.05).Furthermore,ChIP-PCR showed direct interaction between TCF4 and CTGF promoter.Conclusion Our study identifies CTGF as a novel target gene ofβ-catenin-TCF4 signaling pathway,and TCF4 direct targets the promoter of CTGF.