CTP-NPRL2融合蛋白对肾癌原代细胞迁移侵袭能力的抑制及机制研究

CTP-NPRL2 fusion protein inhibits migration and invasion of primary renal cell carcinoma cells

目的将原核表达的氮酶调节因子2(nitrogen permease regulator 2-like,NPRL2)融合蛋白通过胞浆转导肽(cytoplasmic transduction peptide,CTP)转入肾癌原代细胞内,观察其对肾癌原代细胞迁移侵袭的影响,并分析与肾癌上皮间质转化(epithelial-mesenchymal transition,EMT)的关系。方法胶原酶消化法培养肾癌原代细胞;构建原核表达质粒pET15b-CTP-NPRL2和pET15b-NPRL2,表达融合蛋白CTP-NPRL2和NPRL2,并通过Western blot鉴定目的蛋白的表达。将培养成功的原代细胞分为BLANK组、NPRL2组和CTP-NPRL2组。免疫荧光检测目的蛋白的细胞定位;Transwell迁移侵袭实验检测导入NPRL2蛋白后,对肾癌细胞迁移侵袭能力的影响;Real-time PCR检测Raptor、E-钙粘蛋白(E-cadherin)、波形蛋白(Vimentin)、纤粘蛋白(Fibronectin)m RNA表达水平;Western blot检测Raptor、E-cadherin、Vimentin、Fibronectin蛋白表达水平。结果成功培养出肾癌原代细胞并通过鉴定;测序结果及Western blot检测结果显示质粒构建成功并表达出目的蛋白;免疫荧光检测结果显示CTP-NPRL2融合蛋白可以穿过胞膜并定位于胞浆;迁移和侵袭实验显示CTP-NPRL2组肾癌细胞迁移侵袭能力明显下降(P<0.05);Real-time PCR和Western blot检测结果显示CTP-NPRL2组Raptor、Vimentin与Fibronectin的m RNA、蛋白表达水平与NPRL2组和BLANK组相比明显降低(P<0.05),而E-cadherin的mRNA、蛋白表达水平与NPRL2组与BLANK组相比明显升高(P<0.05)。结论 NPRL2可能通过与Raptor相互作用影响mTORC1的活性,进而调节EMT相关蛋白E-cadherin、Vimentin、Fibronectin的表达量,影响肾癌细胞的迁移侵袭能力。

Objective To study the effect of cytoplasmic transduction peptide （CTP）-mediated nitrogen permease regulator 2-like （NPRL2） fusion protein on the migration and invasion of primary renal cell carcinoma （RCC） cells as well as its impact on epithelial-mesenchymal transition （EMT）. Methods Primary RCC cells were cultured from clinically resected renal tumor tissue. Prokaryotic expression plasmids pET15b- CTP-NPRL2 and pET15b-NPRL2 were constructed and transfected into E. coli BL21. The expression of fusion proteins CTP-NPRL2 and NPRL2 was tested by Western blotting. Primary RCC cells were divided into 3 groups CTP-NPRL2, NPRL2 and blank control by treated with CTP-NPRL2, NPRL2 fusion protein solution and PBS, respectively. Cell invasion and migration ability were evaluated by Transwell assay. The mRNA and protein expression levels of raptor, vimentin, （RT-PCR） and Western blotting, respectively. fibronectin and E-cadherin were detected by real-time PCR Results Morphologic observation and immunochemical assay confirmed the successful culture of primary RCC cells derived from patient specimens. After plasmid construction and transfection, the expression of fusion proteins CTP-NPRL2 and NPRL2 were detected by Western blotting. Green fluorescence was only detected in the cytoplasm of CTP-NPRL2 group, suggesting that CTP successfully mediated NPRL2 protein into RCC cells. Compared with NPRL2 and control groups, the invasion and migration ability of primary RCC cells in CTP-NPRL2 group was significantly decreased （P 〈 0. 05 ）. Also, the mRNA and protein expression levels of raptor, vimentin and fibronectin were significantly decreased, while E-cadherin was significantly increased （ P 〈 0. 05 ） in CTP-NPRL2 group. Conclusion CTP-NPRL2 fusion protein inhibits the migration and invasion ability of RCC. This may partly be associated with decreased activity of mTORC1 mediated by interaction between NPRL2 and raptor, consequently regulating the expression of EMT-correlated E-cadherin, vimentin and fibronectin.