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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 investigate the effect of NPRL2 fusion protein on the primary renal cell carcinoma cells by cytoplasmic transduction peptide (CTP) On behalf of the impact of cell migration and invasion, and analysis and renal cell carcinoma epithelial-mesenchymal transition (EMT) relationship. Methods Primary human renal cell carcinoma cells were cultured by collagenase digestion. The prokaryotic expression plasmids pET15b-CTP-NPRL2 and pET15b-NPRL2 were constructed and the fusion proteins CTP-NPRL2 and NPRL2 were expressed. The expression of the target protein was identified by Western blot. Primary cultured cells were divided into BLANK group, NPRL2 group and CTP-NPRL2 group. Transwell migration assay was used to detect the effect of NPRL2 protein on the migration and invasion of renal cell carcinoma cells. Real-time PCR was used to detect the expression of Raptor, E-cadherin and vimentin Vimentin and Fibronectin mRNA expression levels were detected by Western blot. The protein expressions of Raptor, E-cadherin, Vimentin and fibronectin were detected by Western blot. Results The primary cells of renal cell carcinoma were successfully cultured and identified. The sequencing results and Western blot results showed that the plasmid was successfully constructed and expressed the target protein. The results of immunofluorescence showed that CTP-NPRL2 fusion protein could cross the membrane and locate in the cytoplasm ; Migration and invasion experiments showed that the migration and invasion ability of CTP-NPRL2 cells were significantly decreased (P <0.05). The results of Real-time PCR and Western blot showed that the mRNA and protein expressions of Raptor, Vimentin and Fibronectin in CTP- Compared with NPRL2 group and BLANK group, the expression of E-cadherin mRNA and protein in NPRL2 group and BLANK group was significantly lower (P <0.05). Conclusion NPRL2 may affect the activity of mTORC1 through the interaction with Raptor, and then regulate the expressions of EMT-related proteins E-cadherin, Vimentin and Fibronectin, and affect the migration and invasion ability of renal carcinoma cells.