经典瞬时受体电位通道TRPC在肾细胞癌中的功能及调控机制研究

Renal cell carcinoma (RCC) accounts for approximately 90% of all renal malignancies. However, the mechanism by which RCC develops remains largely unknown. Altered intracellular calcium can trigger pathological and physiological changes including cell growth, differentiation and death. These events are implicated in tumor initiation and progression. The transient receptor potential (TRP) protein superfamily is a group of voltage-independent cation-permeable ion channels, most resulting in changed intracellular calcium. These channels have been implicated in several diseases including cancer. In this study, we will investigate the expression profile of TRP canonical (TRPC) channels in radical nephrectomy specimens in order to clarify the clinical significance of the TRPC channels in RCC. We will generate stable RCC cell lines transfected with the plasmid of green fluorescent protein-tagged TRPC or tetracycline-inducible shRNA against TRPC, respectively. We will further subcutaneously inject these cells to the nude mice to detect the tumorigenic abilities. Several molecular approaches will be used, including confocal laser scanning microscopy, whole-cell patch clamp, tumor invasion assay, cell-cycle analysis, In-Cell Western, cell surface protein biotinylation and immunohistochemical staining. Our findings may suggest that altered expression profile of TRPC channels contributes to the development of RCC, and may motivate testing of drugs with the potential to attenuate TRPC-channelopathies which can lead to several diseases including cancer.

肾细胞癌约占肾脏恶性肿瘤的90%，预后较差。然而，肾细胞癌的发生机制尚不清楚。钙离子是重要的第二信使，细胞内钙稳态失衡将直接导致细胞生理及病理功能改变，影响细胞的存活和凋亡。瞬时受体电位(TRP)通道是位于细胞膜上的一类阳离子通道超家族，调控钙离子内流。近来有研究表明，TRP通道和多种疾病密切相关。课题组推测TRP通道在肾细胞癌中具有重要作用。本研究拟应用PCR、免疫印迹及免疫组化等方法检测经典型TRP（TRPC）通道在肾细胞癌组织中的表达，并与癌旁组织进行比较，探讨其临床意义。课题组拟分别建立TRPC过表达的和可诱导TRPC沉默的稳定转染肾癌细胞株，并将其接种于裸小鼠构建移植瘤模型，研究TRPC对肾细胞癌生物学行为的直接影响。该研究综合临床数据、体外细胞实验及实验动物模型，借助成熟的分子生物学手段，拟阐明TRPC在肾细胞癌中的表达及调控机制，为肾细胞癌的治疗提供新思路。

肾细胞癌约占肾脏恶性肿瘤的90%，预后较差。探讨肾细胞癌的发生、发展机制具有重要临床意义。细胞内钙稳态失衡直接导致细胞生理及病理功能改变，影响包括肿瘤在内的各种细胞的存活和凋亡。经典型瞬时受体电位(TRPC)通道精密调控细胞内钙离子水平。近来有研究表明，TRPC通道与包括肿瘤在内的多种疾病密切相关。课题组旨在探索TRPC通道在肾细胞癌中的作用及相关分子调控机制。.本课题临床数据显示，肾细胞癌患者血清表皮生长因子（EGF）、可溶性尿激酶受体（suPAR）蛋白水平显著增高（P<0.05 vs. 健康对照组）。免疫印迹及免疫组化结果显示，与癌旁组织相比，肾细胞癌组织TRPC6通道蛋白表达显著升高（P<0.05 vs. 癌旁组织）。细胞实验及动物实验进一步揭示EGF对TRPC6具有负调控作用，即肾癌患者血清中升高的EGF通过磷酸化ERK，促进TRPC6从细胞膜向细胞浆转移；与此对应的是，suPAR对TRPC6具有正调控作用，即肾癌患者血清中升高的suPAR通过RhoA的介导，促进TRPC6从细胞浆向细胞膜融合。两种调控最终结果是肾细胞癌TRPC6表达显著升高，并伴随钙超载。这可能是肾癌细胞增殖的重要机制之一。.本课题综合临床数据、体外细胞实验及实验动物模型，初步阐明了TRPC在肾细胞癌中的表达及调控机制，为肾细胞癌的治疗提供了新思路。