稳定沉默TRB3细胞模型及TRB3启动子报告基因的建立
Construction of cell model with long-term silencing of TRB3 and TRB3 promoter reporter gene vector
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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。Abstract: 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.
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Keywords:
- TRB3 /
- long-term silence /
- reporter gene /
- tumor /
- migration
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