CDCP1基因重组慢病毒表达载体的构建及其对宫颈癌细胞增殖与迁移的影响

Construction of recombinant lentivirus vector carrying CDCP1 and its effect on the proliferation and migration of cervical cancer cells

目的:构建含CUB结构域包含蛋白1(CUB domain containing protein 1,CDCP1)基因的重组慢病毒表达载体,研究CDCP1蛋白对宫颈癌细胞增殖与迁移的影响。方法:以人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)cDNA为模板,采用PCR法扩增出CDCP1基因,将其克隆至pHAGE-CMV-MCS-IzsGreen载体中,构建重组慢病毒表达载体pHAGE-CDCP1。将重组质粒pHAGE-CDCP1以及对照质粒pHAGE分别与包装质粒psPAX2以及包膜质粒pMD2.G共转染入人胚肾上皮细胞(293 T细胞),48 h后收集病毒悬液,并采用梯度稀释法测定病毒滴度。将重组慢病毒CDCP1感染人宫颈癌HeLa细胞,通过免疫印迹法检测CDCP1蛋白的表达,采用CCK-8实验检测过表达CDCP1对HeLa细胞增殖的影响;采用划痕实验和Transwell细胞迁移实验观察过表达CDCP1对HeLa细胞迁移能力的影响。结果:质粒双酶切鉴定以及核酸测序比对结果证实重组质粒pHAGE-CDCP1构建成功。重组pHAGE-CDCP1慢病毒感染宫颈癌HeLa细胞后细胞状态良好,CDCP1蛋白表达水平明显增加。高表达CDCP1可以显著提高HeLa细胞的增殖和迁移能力。结论:过表达CDCP1能够促进宫颈癌HeLa细胞的增殖和迁移。

Objective:To construct the recombinant lentiviral vector carrying CUB domain containing protein 1(CDCP1)and explore its effect on the proliferation and migration of cervical cancer cells.Methods:The sequence of CDCP1 was cloned from human umbilical vein endothelial cells(HUVECs),amplified by polymerase chain reaction(PCR)and then inserted into lentiviral vector pHAGE-CMV-MCS-IzsGreen.Then recombinant plasmid pHAGE-CDCP1,package plasmid psPAX2 and envelope plasmid pMD2.G were co-transfected into human embryonic kidney epithelial cells(293T cells).Supernatant were harvested and filtered 48 hours later and then the viral titer was determined by gradient dilution.Next,lentivirus-CDCP1 was used to infect HeLa cells,then the CDCP1 protein expression was detected by Western blotting.Cell Counting kit-8 was used to evaluate the effect of CDCP1 on the proliferation of HeLa cells.Transwell migration and wound-healing assays were adopted to detect the effect of CDCP1 overexpression on cell migration.Results:The results of plasmid double enzyme digestion and nucleic acid sequencing confirmed that the recombinant plasmid pHAGE-CDCP1 was successfully constructed.Recombinant lentivirus pHAGE-CDCP1 infected cervical cancer HeLa cells were in a good condition and showed a significantly increased CDCP1 expression.The highly expressed CDCP1 could significantly increase HeLa cell proliferation and migration.Conclusion:Overexpression of CDCP1 could promote the proliferation and migration of cervical cancer HeLa cells.