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.