摘要
构建人TRB3基因的shRNA真核表达载体和稳定沉默TRB3的人结肠癌细胞系HCT-8,构建TRB3启动子区的荧光素酶报告基因,为以TRB3为靶点的药物研究提供有效的筛选平台。针对TRB3的mRNA设计寡核苷酸序列,构建TRB3-shRNA表达载体和control-shRNA阴性对照载体,宿主菌扩增,并测序鉴定。测序正确的重组质粒转染HCT-8细胞,以潮霉素筛选,分别建立稳定表达TRB3-shRNA和control-shRNA的HCT-8细胞系。通过细胞划痕-修复实验检测细胞的迁移能力。同时,通过克隆、酶切、连接、转化和扩增,构建TRB3启动子区的荧光素酶报告基因pTRB3-Luc,转染HEK293ET细胞并给予衣霉素刺激,检测荧光素酶报告基因的活性。经测序证实,TRB3-shRNA真核表达载体构建成功,插入的DNA片段与设计序列完全一致。在建立的稳定表达TRB3-shRNA的HCT-8细胞中,TRB3的mRNA和蛋白表达水平都显著下降,细胞的迁移能力显著降低。同时,TRB3启动子区荧光素酶报告基因pTRB3-Luc构建成功,并在衣霉素诱导下呈现剂量依赖性的活性增强。TRB3-shRNA重组质粒、稳定沉默TRB3的HCT-8细胞系和TRB3启动子报告基因的建立为进一步研究TRB3在肿瘤发生发展过程中的作用以及TRB3抑制剂的筛选提供了有力的工具。
The purpose is to construct human TRB3-shRNA eukaryotic expression vector,human colon cancer cell line HCT-8 with long-term silencing of TRB3,and the luciferase reporter gene vector of TRB3 promoter.Oligonucleotide sequences were designed according to TRB3 mRNA,and TRB3-shRNA expression vector and negative control-shRNA vector were constructed.The qualified recombinant plasmids were transfected into HCT-8,and cells were screened by hygromycin to establish the cell lines expressing TRB3-shRNA and control-shRNA stably.Scratch-restoration experiment was applied to detect the migration ability of cells.Meanwhile,the luciferase reporter gene vector of TRB3 promoter,pTRB3-Luc,was constructed and transfected into HEK293ET cells.Cells were treated with tunicamycin,and the luciferase activity of pTRB3-Luc was detected.DNA sequencing revealed that TRB3-shRNA and control-shRNA eukaryotic expression vectors were constructed successfully.Expression of TRB3 mRNA and protein was down-regulated significantly in HCT-8 cells expressing TRB3-shRNA stably,and the migration ability of TRB3-shRNA cells was weakened significantly.And also,pTRB3-Luc was constructed successfully,and the luciferase activity was enhanced by tunicamycin in a concentration-dependent manner.The construction of TRB3-shRNA recombinant vector,HCT-8 cell lines with long-term silencing of TRB3 and pTRB3-Luc reporter gene will provide potential alternatives for further investigation of the effect of TRB3 on carcinogenesis and tumor progression.
出处
《中国药科大学学报》
CAS
CSCD
北大核心
2012年第2期182-186,共5页
Journal of China Pharmaceutical University
基金
国家自然科学基金资助项目(No.30973557
No.81101595)
中央级公益性科研院所基本科研创新药物发现与新技术专项资助项目(No.2010CHX12)~~
关键词
TRB3
稳定沉默
报告基因
肿瘤
迁移
TRB3
long-term silence
reporter gene
tumor
migration This study was supported by the National Natural Science Foundation of China（No.30973557
No.81101595） and the Basic Research Program of the Institute of Materia Medica（No.2010CHX12）
