Application of Sleeping Beauty transposon in screening lung cancer-related genes

doi:10.3969/j.issn.1009-976X.2023.02.005

Abstract

Abstract: Objective To explore the application of Sleeping Beauty transposon in screening lung cancer related genes. Methods The Sleeping Beauty transposon was used to insert T2/Onc cassette to mutate HBEC （human bronchial epithelial cells）. The positive clones were selected and the CIS （common insertion sites） and candidate genes were screened by high-throughput sequencing. The function of COL11A1 was verified in non-small cell lung cancer （NSCLC） cell line NCI-H1299. CCK-8 cell proliferation and other assays were used to detect the proliferation ability and cisplatin sensitivity of NCI-H1299 cell line before and after knocking down COL11A1. Transwell migration and invasion assays were used to compare the migration and invasion ability of them. Results CIS were determined by Monte Carlo Simulation, which is commonly used in transposon screening. A total of 252 CIS were found with P<0.05 as the bound, including 675 candidate genes. The COL11A1 can promote the proliferation, migration and invasion of non-small cell lung cancer （NSCLC） cell line NCI-H1299 and mediate cisplatin resistance. Conclusion This study demonstrated that the model of mutagenesis of HBEC by SB transposon insertion into T2/Onc can well screen lung cancer-related genes, and confirmed that COL11A1 could promote the proliferation, migration and invasion of NSCLC and mediate cisplatin resistance and epithelial-mesenchymal transition.

Key words: non-small cell lung cancer, Sleeping Beauty transposon, genetic screening, COL11A1

摘要： 目的探讨睡美人（SB）转座子筛选肺癌相关基因的应用。方法采用Sleeping Beauty转座子插入T2/Onc盒子（T2/Onc cassette）诱变人支气管上皮细胞（HBEC）的模型,挑选阳性克隆,高通量测序筛选共同插入位点（CIS）及候选基因。在非小细胞肺癌（NSCLC）细胞系NCI-H1299中验证筛选出的COL11A1基因功能,采用CCK-8细胞增殖实验等检测NCI-H1299细胞系敲低COL11A1前后的增殖能力及其对顺铂的敏感性变化,采用Transwell细胞迁移、侵袭实验比较其迁移、侵袭能力变化。结果使用转座子筛选中通用的蒙特卡洛模拟法确定CIS,以P<0.05为界,共找到252个CIS,包括675个候选基因。结果显示,筛选出的COL11A1基因可以促进NCI-H1299细胞系增殖（P<0.01）、迁移（P<0.001）、侵袭（P<0.001）并介导其顺铂耐药（P<0.001）。结论本研究证明了使用SB转座子插入T2/Onc诱变HBEC的模型能够很好地筛选出肺癌相关基因,并证实了COL11A1可以促进NSCLC细胞增殖、迁移、侵袭并介导顺铂耐药及上皮间质转换。

关键词: 非小细胞肺癌, 睡美人转座子, 基因筛选, COL11A1

CLC Number:

R734

ZHU Xiang-ping, GAO Zeng-hong, MA Ruo-wu, WANG Jin, LIU Ying, GUO Ya-bin. Application of Sleeping Beauty transposon in screening lung cancer-related genes[J]. Lingnan Modern Clinics In Surgery, 2023, 23(02): 118-126.

朱祥平, 高增鸿, 马若骛, 汪瑾, 刘颖, 郭雅彬. 用Sleeping Beauty转座子筛选肺癌相关基因及功能研究[J]. 岭南现代临床外科, 2023, 23(02): 118-126.

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