| t（6；11）染色体易位肾细胞癌中Alpha基因片段的克隆及启动子活性分析 |
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| 引用本文： | 詹鹤琴,袁毅,秦蓉.t（6；11）染色体易位肾细胞癌中Alpha基因片段的克隆及启动子活性分析[J].中国肿瘤临床,2014,41(15):951-956. |
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| 作者姓名： | 詹鹤琴 袁毅 秦蓉 |
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| 作者单位： | ①.安徽医科大学病理学教研室（合肥市 230032） |
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| 基金项目： | 国家自然科学青年基金项目81202006 |
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| 摘 要： | 目的 构建包含不同Alpha基因片段的重组报告质粒和Alpha1-TFEB融合基因表达质粒，并评价Alpha基因启动子活性。 方法 利用软件对Alpha基因进行启动子预测；采用PCR技术扩增出5种不同长度的Alpha基因片段和正常TFEB基因启动子序列pTFEB，构建含Alpha基因片段和pTFEB的重组报告质粒；采用脂质体转染法将其分别转染至人胚肾293T细胞；通过荧光素酶活性检测确定Alpha基因的启动子活性。同时构建含Alpha1-TFEB融合基因表达质粒，转染293T细胞后，采用Western blot法检测转染前后TFEB蛋白的表达水平。 结果 成功构建含不同Alpha基因片段和pTFEB的重组报告质粒。与pGL3-Basic质粒转染组进行比较，Alpha1、Alpha2、Alpha3、Alpha4、Alpha5质粒转染组的荧光素酶活性显著增高（P＜0.01）；与正常TFEB基因启动子进行比较，Alpha1、Alpha2、Alpha5的荧光素酶活性明显增高（P＜0.01）；与pGL3-Enhancer质粒转染组进行比较，pGL3-Enhancer-Alpha1-TFEB表达质粒组TFEB蛋白的表达明显升高。 结论 t（6；11）染色体易位肾细胞癌中Alpha基因具有启动子活性，可促进TFEB表达，Alpha5片段最强活性区域位于643~693位碱基序列。
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| 关 键 词： | Alpha基因 启动子活性 Alpha-TFEB肾肿瘤 t（6 11）染色体易位 |
| 收稿时间： | 2014-01-22 |
Cloning of Alpha gene segments from t(6;11) translocation renal cell carcinoma and analysis of their promoter activities |
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| Affiliation: | ①.Department of Pathology, Anhui Medical University, Hefei 230032 China②.Department of Osteology, Anhui Provincial Children's Hos-pital, Hefei 230051, China |
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| Abstract: | Objective 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 promoter were constructed. Liposome transfection was used to transfect these vectors into the human embryo kidney 293T cells. The promoter activity of the Alpha gene was evaluated via luciferase assay. Meanwhile, the recombinant Alpha1-TFEB plasmid was constructed 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 enhances TFEB expression. The strongest promoter activity region is in Alpha5 with a sequence from 643 bp to 693 bp. |
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