RNAi抑制CAR基因对人肺鳞癌细胞系NCI-H520裸鼠皮下移植瘤生长的影响
Effect of RNA interference on expression of coxsackievirus and adenovirus receptor during human lung squamous cell carcinoma NCI-H520 cell line xenograft growth in nude mice
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摘要:目的 建立RNAi抑制柯萨奇病毒腺病毒受体（coxsackievirus and adenovirus receptor, CAR）基因表达的肺鳞状细胞癌NCI-H520稳定表达细胞系, 将CAR基因沉默的NCI-H520细胞移植于裸鼠体内构建移植瘤模型, 观察裸鼠体内成瘤率及对瘤体生长等方面的影响。方法 将针对CAR mRNA序列设计的小干扰RNA重组质粒分别以脂质体转染至NCI-H520细胞, 并经G418抗性筛选得到稳定细胞系; 用平板克隆形成实验观察RNAi抑制CAR基因表达对NCI-H520细胞增殖的影响; 通过动态观察瘤体的生长, 进一步验证CAR对肺癌的增殖是否有促进作用。结果 筛选出抑制CAR基因表达效果最佳的一组shRNA, 并建立了能稳定而有效抑制CAR基因表达的肺鳞癌NCI-H520细胞系。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, 但其差异无统计学意义（P>0.05）; 且在肿瘤形成实验中, 实验组瘤重显著低于对照组（P < 0.01）。结论 裸鼠肺癌移植瘤模型构建成功, 为今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。Abstract:Objective This work aimed to establish a lung squamous carcinoma cell line in which the coxsackievirus and adenovirus receptor (CAR) gene expression is stably suppressed by ribonucleic acid (RNA) interference. Colony formation experiments and orthotopic tumor model building were employed to detect the correlation between CAR and tumor formation.Methods Three small interfering RNAs (siRNA) targeting the CAR gene were transfected into a lung squamous carcinoma NCI-H520 cell line. G418 was used to select a cell line where CAR gene expression was stably suppressed by RNA interference. The expression levels of CAR mRNA and protein were detected by Western blot in three NCI-H520 cell lines. The cell line where CAR gene expression was most suppressed was selected. Experiments on colony formation and tumorigenesis were employed to determine the effects of CAR gene on the proliferation of NCI-H520 cells after the CAR expression was reduced.Results Three NCI-H520 cell lines wherein CAR gene expression was stably suppressed were obtained. Results of the colony formation assays suggested that the reduced CAR levels were associated with weakened cell proliferation. However, these data did not reach a statistical significance. The CAR-silenced NCI-H520 clones (CAR-) were inhibited in their ability to engraft the virocytes into the nude mice.Conclusions A cell line NCI-H520 in which CAR gene expression was stably and efficiently suppressed was successfully constructed. The RNAi-mediated gene silencing of CAR can significantly affect the growth of NCI-H520 cells in the tumor-bearing nude mice. CAR is expected to become a new target for treating lung cancers.
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