Cloning of Alpha gene segments from t(6;11) translocation renal cell carcinoma and analysis of their promoter activities / 中国肿瘤临床

ABSTRACT

To construct recombinant reporter plasmids containing different Alpha gene segments and Alpha1-TFEB fusion gene and to evaluate the promoter activity of the Alpha gene. Methods:Promoter regions of the Alpha gene were predicted using a software Primer 0.5. Five Alpha gene segments with different lengths and a normal TFEB gene promoter (pTFEB) were amplified via polymerase chain reaction, and recombinant reporter plasmids containing different Alpha gene segments and a normal TFEB gene pro-moter were constructed. Liposome transfection was used to transfect these vectors into the human embryo kidney 293T cells. The pro-moter activity of the Alpha gene was evaluated via luciferase assay. Meanwhile, the recombinant Alpha1-TFEB plasmid was construct-ed and transfected into the 293T cells. The TFEB expression of the recombinant Alpha1-TFEB plasmid was then detected via Western blot. Results: Recombinant reporter plasmids containing different Alpha gene segments and pTFEB were constructed successfully. Compared with the luciferase activity of pGL3-Basic, that of the groups with Alpha1, Alpha2, Alpha3, Alpha4 and Alpha5 significantly increased (P<0.01). The luciferase activity also increased significantly in the groups with Alpha1, Alpha2 and Alpha5 compared with that of the pTFEB group (P<0.01). The TFEB expression of the pGL3-Enhancer-Alpha1-TFEB was significantly higher compared with that of the pGL3-Enhancer group. Conclusion:In t(6;11) translocation RCC, the Alpha gene has a strong promoter activity and it en-hances TFEB expression. The strongest promoter activity region is in Alpha5 with a sequence from 643 bp to 693 bp.