Effect Of TMED3 On Cell Viability,Migration And Apoptosis In Human Chordoma Cells

Background and objective:Chordoma is a rare bone tumor that occurs in the axial bone.TMED3(Transmembrane Emp24 Protein Transport Domain Containing 3,TMED3)is a novel gene regarded as a regulator in oncogenesis,cancer development and metastasis,but its role in chordoma remains unclear.This study aims to explore the role of TMED3 in the growth of chordoma and explore the underlying molecular mechanisms.Methods:In this study,immunohistochemical staining was firstly performed to preliminarily identify the expression of TMED3 using human chordoma tissue specimen.Then,based on MUG-Chorl and U-CH1 chordoma cells,RT-qPCR and western blot assays were performed to detect the expression level of TMED3 in chordoma cells,which is normalized to the GAPDH gene expression.Lentiviral transfection method was used to knockdown the TMED3 gene in chordoma.Efficiency of lentiviral transfection was evaluated and the expression of TMED3 was determined via qPCR and western blot assays in order to achieve more than 50%knockdown efficiency.Chordoma cells were divided into TMED3-knockdown group(shTMED3 group)and control group(shCtrl group).MTT method,wound-healing assay and transwell assay,PI staining method,AnnexinV-APC staining method were used to respectively detect cell viability,cell migration,cell cycle and apoptosis of chordoma in vitro.Moreover,human apoptotic protein antibody assay and western blot were performed to respectively detect the differential expression of apoptosis-related genes and phosphorylation-related genes.Finally,MUG-Chorl cells with different expression levels of TMED3 were inoculated subcutaneously in nude mice to construct a nude mice xenograft model to verify the effect of TMED3 on chordoma growth in vivo.Results:TMED3 was expressed at a medium to high level in chordoma tissues and cells.The transfection efficiency of lentivirus transfected chordoma cells reached more than 80%,and the knockdown efficiency of TMED3 gene reached more than 50%,which achieved the expected effect.Knockdown of TMED3 significantly inhibited the cell viability,cell cycle and migration ability of chordoma,and promoted cell apoptosis.The apoptosis antibody assay showed that knockdown of TMED3 significantly inhibited the expression of Bcl-2,HSP27,IGF-I,IGF-II,IGFBP-2,and Livin.Besides,western blot experiments showed that the expression of phosphorylation-related genes,including Akt,CDK6,and CyclinDl were down-regulated,while MAPK9 was significantly up-regulated.In addition,the results of the xenograft model showed that the volume and quality of chordomas in the shTMED3 group were significantly reduced compared with the control group.After TMED3 knockdown,the staining rate of Ki67 in chordoma tissue was significantly reduced,proving that the expression of TMED3 can significantly promote chordoma growth in vivo.Conclusion:Present study shows that TMED3 can promote chordoma cell viability,cell migration and inhibit apoptosis,suggesting that TMED3 play a positive role on chordoma growth and may be a new potential target for chordoma treatment.