Mechanism Of TEF3-1 And UBQLN2 On Tumorigenesis

At present, the study and treatment of malignant tumors has reached the genetic level, through the study of tumor-related genes can be applied to malignant tumor targeted therapy. TEF3-1 is proposed to be key mediator of processes such as proliferation, migration and transformation, which are important for tumorigenesis. UBQLN2 is commonly known as a pathogenic gene of ALS, but has also been implicated in the proliferation of several malignancies. UBQLN2 immunocytochemisyry may represent a clinicopathologicall marker in tumor grade and stage of malignanies.AH 109 cells cannot grow on plates lacking Trp, Leu, and His, providing selective markers for yeast two-hybrid assay in which the enzyme for Trp synthesis is expressed from pGBKT7-based constructs while the gene encoding the enzyme for His synthesis and a p-galactosidase gene (MEL1) are expressed from two independent UAS-containing basal promoters (usually silent) in the AH 109 cells. The UAS sequences provide binding site for Gal4 DNA-BD, which is also expressed from the pGBKT7-based constructs. When a protein or protein domain capable of activating gene expression is brought to the proximity of the UAS-containing promoters through interaction between the UAS and the Gal4 DNA-BD, the transformed AH109 cells gain ability to grow on SD/-Trp/-His medium and produce blue pigments with X-gal staining. Then expression the different gene with the plasmid pGBKT7 in AH 109 yeast cells maybe the simple and straight method to determine the transcriptional domains of the gene. Taking advantage of the efficient reporter design of yeast two-hybrid system, we characterized the TEF3-1 domains in activating gene expression. Previously study usually mentioned that the C-terminal domain of TEF3-1 has the transcriptional activity, however, our data shows that the peptides TEF3-l1-66 and TEF3-1197-434 functioned as two independent activation domains, suggesting that N-terminal domain of TEF3-1 also has transcriptional activation capacity. Additionally, more deletions of amino acids 197-434 showed that only the peptides TEF3-1197-265 contained the minimum sequences for the C-terminal transcriptional activation domain. The protein structure is predicted to contain a helix-turn-helix structure in TEF3-l1-66 and four ? sheets in TEF3-1197.265-These structures were indispensable for the function of the domains. Finally, after the truncated fragments of TEF3-1 were expressed in HUVEC cells, the whole TEF3-1 and the two activation domains could increase F-actin stress fiber. Further analysis and characterization of the activation domains in TEF3-1 may broaden our understanding of the gene involved in angiogenesis and other pathological processes, and the mechanism of overexpression as a clinicopathological marker.In a study of another tumor-associated gene, UBQLN2, that could also serve as a biomarker in some tumors. Taking advantage of the powerful gene editing tool CRISPR/Cas9 system, we obtained the UBQLN2 knockout HeLa cell lines. The results of identifications of UBQLN2 expression and UBQLN2 mutations by western blot and sequencing respectively showed that we successfully established UBQLN2 knockout HeLa cells. Further study showed that UBQLN2 gene can mediated the proliferation of HeLa cells, independently form cell cycle. Interestingly, in the MG132-treated HeLa cells, the cell viability of UBQLN2 knockout cells was significantly higher than that of wild-type HeLa cells. The increase of cell viability was result in both the reduction of apoptosis and the change of proportion of G2/M in the cell cycle. TALEN system was employed for construction of Ubqln2 knockout rat lines, which identificated by western blot and sequencing. Behavioral analysis revealed that cognitive level was increased in Ubqln2 knockout rats, suggesting that Ubqln2 knockout has a protective effect on neurons. We used the CRISPR/Cas9 and TALEN to established UBQLN2 knockout HeLa cells and rats respectively. Establishment of in vitro and in vivo models had important practicable value for study on the mechanism of UBQLN2 gene in tumorigenesis.