【摘要】 背景与目的小G蛋白家族成员Rap2a可调控内皮素和细胞粘附从而影响细胞运动及细胞与基质间相互作用,但其在肿瘤发生发展中的作用仍属未知。克隆人Ras家族小G蛋白Rap2a的cDNA,构建其真核表达质粒并在肺癌细胞表达,初步探讨Rap2a在肺癌发生发展中的作用。方法 Western blot检测Rap2a在肺癌细胞中的内源性表达。人骨肉瘤细胞株U2OS提取细胞总RNA,经逆转录聚合酶链式反应逆转录成cDNA,PCR扩增Rap2a基因,酶切后插入pcDNA3.1(+)构建真核表达质粒pcDNA3.1(+)-Rap2a,采用酶切及测序鉴定。重组质粒转染H1299和A549细胞,Western blot检测目的基因表达。Transwell小室迁移实验观察Rap2a对肺癌细胞迁移能力的影响。明胶酶谱实验检测Rap2a对细胞分泌基质金属蛋白酶(matrix metalloproteinase,MMP)2的影响。结果与正常细胞相比,肺癌细胞中Rap2a基础表达水平明显增高。双酶切及测序结果显示重组质粒pcDNA3.1(+)-Rap2a成功构建,Werstern blot检测到H1299和A549细胞有相应蛋白表达。迁移实验结果显示转染Rap2a基因后肿瘤细胞迁移能力明显增加。明胶酶谱实验结果显示Rap2a过表达后肺癌细胞分泌MMP2的量随之增加。结论人Rap2a真核表达质粒成功构建,Rap2a基因在肺癌细胞株成功表达并能促进肺癌细胞的迁移能力。更多还原

【Abstract】 Background and objective Rap2 a, a member of the small GTPase superfamily, plays a critical role in regulating the function of integrin and cell adhesion, thereby controlling cell motility and cell/matrix interactions. However, the function of Rap2 a in carcinogenesis is still poorly understood. To clone Rap2 a cDNA, which belongs to human Rasrelated small G protein superfamily, we constructed its eukaryotic expression vector and determined its expression in lung cancer cells. The aim of this study is to explore the role of Rap2 a in carcinogenesis. Methods The levels of endogenous Rap2 a protein in lung cancer cells were measured by Western blot. Total RNA of human osteosarcoma cells U2 OS was extracted and reverse-transcribed into cDNA by RT-PCR. Then, Rap2 a gene was amplified by PCR and inserted into pcDNA3.1(+). The reconstructed plasmid was identified by restricted enzyme digestion and sequencing. pcDNA3.1(+)-Rap2 a was transfected into H1299 and A549 cells, the expression of Rap2 a was detected by Western blot. In addition, the migratory abilities of lung cancer cells were evaluated by Transwell assay. Matrix metalloproteinase(MMP)2 enzyme activity was evaluated by gelatin zymography. Results Rap2 a is significantly upregulated in lung cancer cells. The results of enzyme digestion and sequencing showed that the coding sequence of pcDNA3.1(+)-Rap2 a was right and was inserted into the vector correctly. The results of Western blot showed that H1299 and A549 cells were transfected successfully. Transwell assay indicated that the ectopic expression of Rap2 a promotes lung cancer cells migration. Correspondly, enzyme activity of MMP2 also increased. Conclusion Eukaryoticexpression plasmid pcDNA3.1(+)-Rap2 a was constructed successfully. Rap2 a could be expressed in lung cancer cells efficiently and promotes lung cancer cell migration.更多还原