融合基因Alpha-TFEB编码产物TFEB在t(6;11)易位肾细胞癌的作用和机制研究

Renal cell carcinoma (RCC) with t(6;11) translocation is a newly identified malignant neoplastic entity. The chromosomal translocation of t(6;11) can form Alpha-TFEB fusion gene, resulting in overexpression of native TFEB protein. Recently, we reported the first Chinese patient with this entity of RCC. Our results demonstrated that TFEB protein encoded by Alpha-TFEB fusion gene was overexpressed in this kind of RCC, whereas it was not detectable in the normal renal tissue. The results suggest that the overexpression of TFEB may be correlated with tumorigenesis of RCC with t(6;11) translocation. At present, it is unclear about the role and mechanism of TFEB in this kind of RCC, and whether TFEB protein can be potential target point in the treatment of RCC with t(6;11) translocation. On basis of previous research results, we will explore and reveal the effect of TFEB encoded by Alpha-TFEB gene on the cell proliferation, colony formation, cell apoptosis and invasive power of immortalized human embryonic kidney cell and renal cell carcinoma cell, and nude mice model will be constructed in order to investigate the tumorigenicity of the transfected cells with steady expression of TFEB in nude mice and its effect on growth and metastasis of renal cell carcinoma in vivo. Meanwhile, we also investigate whether TFEB promotes tumorigenesis through upregulating BCL-2 expression. This study will be helpful to deeply understand the role and mechanism of t(6;11) translocation in the genesis of RCC, and may provide a new candidate target for the treatment of this entity of RCC.

t(6;11)易位肾细胞癌是一类新认识的恶性肿瘤。t(6;11)染色体易位可形成Alpha-TFEB融合基因，导致TFEB蛋白的过表达。最近，我们首次报道了中国人t(6;11)易位肾癌病例。研究结果显示该肿瘤细胞过表达Alpha-TFEB融合基因编码产物TFEB，但正常肾组织并不表达，提示TFEB过表达可能与该肿瘤的发生有关。目前关于TFEB过表达在该肿瘤中的作用和机制及TFEB能否成为潜在治疗靶点尚不清楚。本课题拟在前期研究基础上，探索和揭示TFEB对人胚肾细胞及肾癌细胞增殖、克隆形成、凋亡及侵袭力的影响；并建立裸鼠模型，在体观察稳定表达TFEB阳性细胞在裸鼠体内的成瘤性及对肾癌生长或转移的影响；同时探讨TFEB是否通过上调BCL-2的表达而促进肿瘤的发生。本研究结果将有助于深入认识t(6;11)易位在肾癌发生中的作用和机制，可能为治疗提供新的候选靶点。

t(6;11)易位肾细胞癌是一类新认识的恶性肿瘤。t(6;11)染色体易位可形成Alpha-TFEB融合基因，导致TFEB蛋白的过表达。我们于2010年首次报道了中国人t(6;11)易位肾癌病例。前期研究结果显示该肿瘤细胞过表达Alpha-TFEB融合基因编码产物TFEB，但正常肾组织并不表达，提示TFEB过表达可能与该肿瘤的发生有关。目前关于TFEB过表达在该肿瘤中的作用和机制及TFEB能否成为潜在治疗靶点尚不清楚。本课题主要内容为构建了人Alpha-TFEB融合基因真核表达质粒，筛选并鉴定出稳定表达TFEB的阳性细胞；在细胞水平探讨了Alpha-TFEB编码的TFEB蛋白对人胚肾上皮细胞HK-2及肾细胞癌细胞CaKi-2增殖、克隆形成、凋亡及侵袭力的影响；在动物实验水平探索了稳定表达TFEB阳性细胞在裸鼠体内的成瘤性及对肾癌细胞生长或转移的影响；探索TFEB是否通过上调BCL-2的表达而促进肿瘤的发生。本研究结果显示Alpha-TFEB融合基因中Alpha基因具有启动子活性，且其启动子活性最强的区域位于含有第二个启动子预测位点的Alpha5片段（617-755）。成功构建了pLVX-Puro-Alpha-TFEB慢病毒表达载体，并筛选到稳定表达TFEB的HK-2-Alpha-TFEB和CaKi-2-Alpha-TFEB细胞。我们的结果显示稳定表达TFEB的HK-2-Alpha-TFEB细胞和CaKi-2- Alpha-TFEB细胞组的增殖能力、侵袭力、克隆形成率及抗凋亡能力明显高于未转染TFEB的HK-2细胞和CaKi-2细胞组；成功建立裸鼠模型，接种HK-2细胞组、无血清培养基组的裸鼠均未见肿瘤形成；而接种HK-2-Alpha-TFEB细胞组、CaKi-2细胞组、CaKi-2- Alpha-TFEB细胞组均见肿瘤形成，且接种HK-2-Alpha-TFEB细胞组、CaKi-2- Alpha-TFEB细胞组形成的肿瘤明显大于CaKi-2细胞组。稳定表达TFEB的HK-2-Alpha-TFEB和CaKi-2-Alpha-TFEB细胞组中BCL-2的表达明显高于未转染TFEB的HK-2细胞和CaKi-2细胞组。本研究结果有助于深入认识t(6;11)易位在肾癌发生中的作用和机制，为其治疗提供新的候选靶点和理论依据，具有重要的科学意义及广阔的应用前景。