摘要
目的本研究旨在调查通过siRNA抑制FHL2表达后对β-catenin活性及其下游基因sur-vivin表达的影响,探讨两者在肠癌发生中的协同作用。方法 (1)PCR扩增含有β-catenin作用位点、长为340bp的survivin基因启动子片段,克隆入PGL3-basic载体,构建荧光素酶报告质粒pluc340。(2)脂质体瞬时转染法将β-catenin活性报告质粒pTOPFLASH、pFOPFLASH和pluc340转染入胃肠癌及正常细胞株,双荧光素酶法测定其启动子活性,并评价其对fhl2siRNA的反应。(3)RT-PCR方法检测基因表达。结果成功构建了survivin启动子片段pluc340;荧光素酶活性检测显示:β-catenin转录活性在胃肠癌细胞明显高于正常细胞;siRNA抑制FHL2表达后,β-catenin转录活性下降,survivin表达降低。结论抑制FHL2的表达能够降低β-catenin转录活性及下游癌基因的表达,干扰Wnt信号通路,说明fhl2有望成为肠癌治疗的新靶点。
Objective It is reported that FHL2 is able to interact and then promote the transcriptional activity of β-catenin.The aim of this study is to evaluate the effect of fhl2 siRNA on the transactivities of β-catenin and its downstream gene,survivin.Methods （1）survivinfragment amplified by PCR was inserted into PGL-basic vector to establish recombinant plasmid named as pluc340 which contained luciferase promoter at the downstream.（2）pTOPFLASH,pFOPFLASH,siRNA and pluc340 were transfected into gastrointestinal cells to detect their transactivities by dual-luciferase assay.（3）mRNA expressions were detected by RT-PCR.Results survivin promoter was established successfully.The transactivity of β-catenin was significantly higher than that in the normal cells.The transcriptional activities of β-catenin and survivin were suppressed by fhl2 siRNA.Conclusion The results indicated that FHL2inhibition was capable of suppressing the transcriptional activity of β-catenin and thus interfering the process of Wnt signaling pathway.Therefore,fhl2 might be a promising target for innovative treatment of colon cancer.
出处
《肿瘤防治研究》
CAS
CSCD
北大核心
2010年第9期992-995,共4页
Cancer Research on Prevention and Treatment
