Construction And Identification Of Trb3-shRNA Eukaryotic Expression Vector And Its Effects On The Apoptosis And Proliferation Of Human Tongue Squamous Carcinoma Cells

Objective:To construct and identify the human Trb3-shRNA eukaryotic expression vector and Trb3 knockdown tongue squamous cell carcinoma line Tca8113, observe its effect on inhibiting expression of target gene, assess the effects of knockdown of Trb3 on proliferation and apoptosis, and explore the potential mechanisms.Methods:Molecular cloning were performed for constructing the Trb3-shRNA eukaryotic expression vector:pSUPER-shTrb3 and Trb3 stable knockdown cell lines; Real-time PCR and Western Blot were employed to detected the expression of Trb3 mRNA and protein in stable cell lines; Flow cytometry were used to analyze tumor cell's proliferation and apoptosis; the biomarker of apoptosis were assessed by Western Blot.Results:The results of the restriction enzyme digestion and DNA sequencing demonstrated that the inserted sequences were exactly correct. The Trb3-shRNA eukaryotic expression vector:pSUPER-shTrb3 and Trb3 stable knockdown cell lines were constructed successfully. The expression of Trb3 mRNA and protein in Trb3 stable knockdown cell lines were dramatically down-regulated compared with the control group (P<0.05). Flow cytometry observed that knockdown Trb3 promoted cells'proliferation and inhibited apoptosis under endoplasmic reticulum stress (ERS), and also the biomarker of apoptosis (cleaved-caspase3,7, cleaved-PARP) were depression.Conclusion:Trb3 is involved in regulating proliferation and apoptosis of Tongue squamous carcinoma cell lines Tca8113 under ERS. Construction of this cell model provides the underlying basis for therapy and research targeted Trb3 gene.