应用以人转录组为靶点的shRNA文库筛选肺癌肿瘤相关抑制基因

Screening of lung cancer tumor-associated suppressor genes using shRNA library targeting human transcriptome

目的应用shRNA文库筛选肺上皮细胞相关肿瘤抑制基因,并验证其抑制细胞恶性转化的功能,为肿瘤防治提供新的靶点。方法用GP2-293病毒包装细胞系建立shRNA逆转录病毒文库,感染永生化肺上皮细胞BEAS-2B,经软琼脂克隆形成实验筛选转染的上皮细胞克隆,选择转化细胞克隆经PCR扩增出所插入的shRNA片段,经Sanger法测序和比对确定相应shRNA的靶点基因。通过软琼脂克隆及裸鼠成瘤实验验证候选肿瘤抑制基因INPP4B的肿瘤抑制功能。MTT实验检测细胞的增殖情况。结果通过软琼脂克隆形成筛选出6个候选基因,选取INPP4B进行功能验证。在BEAS-2B细胞中沉默INPP4B基因可促进细胞的克隆形成,细胞增殖速度加快,沉默细胞系在裸鼠皮下成瘤能力增强,说明INPP4B参与肿瘤形成。结论 shRNA文库及软琼脂克隆形成实验是筛选肿瘤抑制基因的一个有力工具,INPP4B是肺上皮细胞恶性转化抑制因子。

Objective The shRNA library was used to screen the tumor suppressor genes related to lung epithelial cells,and its function of inhibiting malignant transformation in lung cells was preliminarily verified,which provided a theoretical basis and a new therapeutic target for tumor prevention and treatment.Methods The shRNA retrovirus library was constructed by GP2-293 virus packaging cell line to infect the immortalized lung bronchial epithelial BEAS-2B cells.The transfected epithelial cell clones were screened by soft agar colony formation,and a single transformed cell clone with a diameter greater than 1.0 mm was selected.The inserted shRNA fragment was amplified by PCR,and the target candidate gene corresponding to shRNA was determined by the conventional DNA sequencing and blast alignment.A candidate tumor suppressor gene INPP4B was verified by soft agar cloning and tumor formation in nude mice.MTT assay was used to detect the cell proliferation.Results Six lung epithelial malignant transformation inhibitory factors were screened by soft agar colony formation.The candidate INPP4B gene was selected for functional experiments.Silencing INPP4B gene in BEAS-2B cells promoted the formation of clones in the soft agar plates,and the cell proliferation rate was accelerated.The silencing cell line showed the enhanced tumorigenicity in nude mice,indicating that INPP4B was involved in tumor formation.Conclusion shRNA library and soft agar colony formation assays are a powerful tool for screening tumor suppressor genes,and INPP4B is a malignant transformation inhibitor of lung epithelial cells.