天津医药

天津医药 ›› 2020, Vol. 48 ›› Issue (5): 364-369.doi: 10.11958/20192473

• 细胞与分子生物学 • 上一篇    下一篇

EBF1-shRNA对肺腺癌A549细胞体内外增殖能力的影响

王琳，李丁，秦婷婷，任丽△
  

  1. 天津医科大学肿瘤医院检验科；国家肿瘤临床医学研究中心；天津市“肿瘤防治”重点实验室；天津市恶性肿瘤临床医学研究中
    心（邮编    300060）
  • 收稿日期:2019-08-13 修回日期:2019-12-16 出版日期:2020-05-15 发布日期:2020-06-24
  • 作者简介:王琳（1987），女，博士，讲师，主要从事肺癌相关研究

Effect of shRNA-mediated silencing EBF1 gene expression on the proliferation of lung cancer cell line A549 in vitro and in vivo

WANG Lin, LI Ding, QIN Ting-ting, REN Li△ #br#   

  1. Department of Laboratory, Tianjin Medical University Cancer Institute and Hospital; National Clinical Research
    Center for Cancer; Key Laboratory of Cancer Prevention and Therapy, Tianjin; Tianjin    ’s Clinical Research
    Center for Cancer    ；Tianjin 300060, China

  • Received:2019-08-13 Revised:2019-12-16 Published:2020-05-15 Online:2020-06-24

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摘要： 目的 研究EBF1基因下调对肺腺癌A549细胞体内外增殖的影响，并初步探讨其机制。方法 通过免疫组化和RT-PCR检测EBF1在肺癌组织和肺癌细胞系中的表达情况。根据本实验室设计的针对EBF1的小干扰RNA(shRNA)序列，选择一条已经证实最有效的序列作为靶序列和一条随机序列作为阴性对照，构建重组逆转录病毒shRNA表达载体并将其转染入A549细胞中；应用RT-PCR和蛋白质印迹法(Western blot)检测对EBF1基因的沉默效果；应用MTT比色法及BrdU染色法实验检测EBF1-shRNA对A549细胞体外增殖的影响；利用Transwell实验及划痕实验检测EBF1-shRNA对A549细胞体外侵袭及迁移能力的影响；利用流式细胞仪分析各组细胞的细胞周期；通过裸鼠腋窝皮下接种各组细胞，观察EBF1-shRNA对A549细胞在裸鼠体内的致瘤能力的影响；Western blot检测CDK6、P21、P27蛋白表达。结果 EBF1在癌旁组织及肺癌组织中的基质细胞中不表达，在NSCLC及SCLC癌细胞细胞核中表达。EBF1-shRNA有效地沉默了EBF1基因mRNA和蛋白的表达；沉默EBF1基因的表达能够抑制A549细胞的体内外增殖能力，使实验组细胞的细胞周期阻滞于G 1 期；沉默EBF1基因的表达后，实验组细胞中CDK6蛋白表达降低，P21、P27蛋白增加。结论 EBF1-shRNA通过使细胞周期阻滞于G 1 期来抑制肺腺癌A549的体内外增殖能力，这种抑制作用涉及CDK6表达下降以及P21/P27的表达上调。

关键词: 肺肿瘤, 腺癌, RNA干扰, 细胞增殖, 细胞凋亡, 细胞周期, A549细胞, EBF1

Abstract: Objective: To study the effect of EBF1 gene knockdown on proliferation of lung adenocarcinoma A549 cells in vitro and in vivo, and to explore its mechanism.Methods: Immunohistochemistry and RT-PCR were used to detect the expression of EBF1 in lung cancer tissues and lung cancer cell lines. A recombinant retroviral shRNA expression vector was constructed and transfected into A549 cells according to the shRNA sequence of EBF1 designed by our lab. Knockdown of EBF1 gene was detected by RT-PCR and Western blot. MTT and BrdU staining were used to detect the effect of EF1-shRNA on the proliferation of A549 cells in vitro; the detection of the effect of ebf1-shRNA on the invasion and migration of A549 cells in vitro by Transwell and scratch experiments;flow cytometry was used to analyze the cell cycle of each group; subcutaneous inoculation of cells in axilla of nude mice was used to observe the effect of EBF1-shRNA on the tumorigenicity of A549 cells in nude mice; Western blot was used to detect the expression of CDK6, P21 and P27 proteins. Results: EBF1 was not expressed in stromal cells of adjacent tissues and lung cancer tissues, but in nuclei of NSCLC and SCLC cancer cells. EBF1-shRNA knockdown EBF1 gene expression effectively; knockdown EBF1 gene expression can inhibit the proliferation of A549 cells in vitro and in vivo, and block the cell cycle of experimental group at G1 phase; after knockdown EBF1 gene expression, CDK6 protein expression in experimental group cells decreases, while P21 and P27 protein increase. Conclusion: EBF1-shRNA can inhibit the proliferation of lung adenocarcinoma A549 in vitro and in vivo by blocking cell cycle in G1 phase, which involves the decrease of CDK6 expression and the up-regulation of P21/P27 expression.

Key words: lung neoplasms, adenocarcinoma, RNA interference, cell proliferation, cell cycle, A549 cells, EBF1