柯萨奇病毒腺病毒受体在肺癌中的表达和意义

摘要

目的:明确柯萨奇病毒腺病毒受体(CAR)在正常肺组织、癌旁组织及肺癌组织中的表达情况以及在体外CAR失表达对人肺鳞状细胞癌NCI-H520细胞增殖与侵袭迁移的影响.方法:收集肺癌手术切除新鲜标本100例及其对应的癌旁组织和正常肺组织,应用免疫组织化学、Western blot和RT-PCR法检测CAR蛋白和mRNA的表达情况;在体外运用RNAi技术建立稳定的低表达CAR的人肺鳞状细胞癌NCI-H520细胞系,用平板克隆形成实验和Transwell侵袭及迁移实验观察RNAi抑制CAR基因表达对NCI-H520肺癌细胞增殖与侵袭迁移的影响,并通过建立动物模型动态观察瘤体的生长.结果:免疫组织化学结果显示,CAR阳性率为48%.Western blot法检测CAR蛋白和RT-PCR检测CAR mRNA在肺癌组织中明显高于正常肺组织和癌旁组织.筛选得到三组CAR基因表达受到抑制的NCI-H520稳定细胞系.半定量RT-PCR和Western blot结果显示,三组稳定细胞系中CAR mRNA和蛋白质表达均有不同程度的下降,其中shRNA-2抑制作用最强.平板克隆形成实验显示,shRNA-2抑制CAR基因表达后,NCI-H520细胞的增殖能力有所下降(P>0.05),Transwell侵袭及迁移实验显示,NCI-H520细胞侵袭迁移能力明显下降(P<0.05).动物模型结果显示,抑制CAR的表达,则动物体内移植瘤的生长就会缓慢.结论:CAR与肺癌形成和进展有关,并能够促进肺癌细胞的侵袭和迁移活动.裸鼠肺癌移植瘤模型构建成功,为以后肺癌的研究奠定基础.利用RNAi有效抑制了CAR基因的表达,并抑制NCI-H520细胞在裸鼠体内瘤体的生长,CAR有望成为肺癌基因治疗的靶点之一.%Objective To investigate the expression of (Coxsackie virus adenovirus receptor,CAR) in nor-mal lung tissue, para-cancerous tissue and lung cancer tissues and the effect of CAR expression in vitro on pro-liferation,invasion and migration of human lung squamous cell carcinoma NCI-H520 cells. Methods Total 100 cases of lung cancer, adjacent tissues and normal lung tissues were collected. Immunohistochemistry, West-ern blot and RT-PCR were used to detect the expression of CAR protein and mRNA. RNAi technique was used to diminish CAR expression. The effects of RNAi on the proliferation, invasion and migration of NCI-H520 lung cancer cells were studied by plate cloning assay and transwell invasion and migration experiments. The animal model was used to study the growth of the cancer cells in vivo. Results Immunohistochemical results showed gene were screened. Semi-quantitative RT-PCR and Western blot showed that the expression of CAR mRNA and protein in these cells decreased in different degrees, and shRNA-2 had the strongest inhibitory effect. Plate clone formation assay showed that shRNA-2 inhibited the proliferation of NCI-H520 cells (P<0.05). Transwell invasion and migration experiments showed that CAR-inhibited NCI-H520 cells had a significant decrease in in-vasion and migration (P< 0.05). Animal model results show that following the inhibition of CAR expression, the growth of transplanted tumors in animals was inhibited slow. Conclusion CAR is associated with the develop-ment and progression of lung cancer and can promote the invasion and migration of lung cancer cells. Nude mice lung cancer transplanted tumor model was successfully established for the future study of lung cancer. The expression of CAR gene was effectively inhibited by RNAi, and, therefore, the growth of NCI-H520 cells in nude mice was inhibited. CAR was expected to be one of the targets of lung cancer gene therapy.