Screening of lung cancer tumor-associated suppressor genes using shRNA library targeting human transcriptome
10. 11904/j. issn. 1002-3070. 2019. 02. 001
- VernacularTitle: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
- Author:
Hui XU
1
,
2
;
Xi LI
;
Chunshui ZHOU
Author Information
1. 哈尔滨医科大学附属肿瘤医院检验科(哈尔滨 150081)
2. 大连医科大学附属第一医院检验科
- Keywords:
shRNA library;
Soft agar assay;
Tumor suppressor;
Inositol polyphosphate 4-phosphatase type II(INPP4B)
- From:
Practical Oncology Journal
2019;33(2):97-102
- CountryChina
- Language:Chinese
-
Abstract:
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 in-serted shRNA fragment was amplified by PCR,and the target candidate gene corresponding to shRNA was determined by the conven-tional 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 in-hibitory factors were screened by soft agar colony formation. The candidate INPP4B gene was selected for functional experiments. Si-lencing 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.
