正常肺上皮细胞致瘤性转化中抑癌基因的全基因组筛选及数据库验证

赵斌瑜; 张俊磊; 王加琪; 阮艳; 张月; 刘恋恋; 蹇锐; 熊加祥; 姚忠祥

doi:10.16016/j.2097-0927.202112095

您当前的位置：

首页 >

文章列表页 >

正常肺上皮细胞致瘤性转化中抑癌基因的全基因组筛选及数据库验证

基础医学 | 更新时间：2026-01-09

- 正常肺上皮细胞致瘤性转化中抑癌基因的全基因组筛选及数据库验证
- Genome-wide screening of tumor suppressor genes in oncogenic transformation of normal lung epithelial cells and preliminary validation in sample databases
- 陆军军医大学学报 2022年44卷第6期页码：503-512
- 作者机构：
  
  1.400038 重庆，陆军军医大学(第三军医大学)基础医学院：生理学教研室
  2.400038 重庆，陆军军医大学(第三军医大学)基础医学院：组织学与胚胎学教研室
  3.400038 重庆，陆军军医大学(第三军医大学)基础医学院：教学实验中心
  4.400038 重庆，陆军军医大学(第三军医大学)第一附属医院眼科
- 作者简介：
  
  [姚忠祥，E-mail：yaozhx@yahoo.com YAO Zhongxiang, E-mail: yaozhx@yahoo.com
- 基金信息：
  
  国家自然科学基金面上项目(32071014)
- DOI：10.16016/j.2097-0927.202112095
  中图分类号： R394-33;R394.3;R734.2
- 收稿：2021-12-13，
  
  修回：2022-01-04，
  
  纸质出版：2022-03-30
- 稿件说明：
移动端阅览
赵斌瑜, 张俊磊, 王加琪, 等. 正常肺上皮细胞致瘤性转化中抑癌基因的全基因组筛选及数据库验证[J]. 陆军军医大学学报, 2022,44(6):503-512.

Binyu ZHAO, Junlei ZHANG, Jiaqi WANG, et al. Genome-wide screening of tumor suppressor genes in oncogenic transformation of normal lung epithelial cells and preliminary validation in sample databases[J]. Journal of Army Medical University, 2022, 44(6): 503-512.
赵斌瑜, 张俊磊, 王加琪, 等. 正常肺上皮细胞致瘤性转化中抑癌基因的全基因组筛选及数据库验证[J]. 陆军军医大学学报, 2022,44(6):503-512. DOI： 10.16016/j.2097-0927.202112095.

Binyu ZHAO, Junlei ZHANG, Jiaqi WANG, et al. Genome-wide screening of tumor suppressor genes in oncogenic transformation of normal lung epithelial cells and preliminary validation in sample databases[J]. Journal of Army Medical University, 2022, 44(6): 503-512. DOI： 10.16016/j.2097-0927.202112095.

摘要

目的

利用

CRISPR

Cas

9敲除文库在人正常肺上皮细胞致瘤性转化过程中进行全基因组筛选，以发现新的人肺癌抑癌基因，并利用临床非小细胞肺癌(non-small cell lung cancer

NSCLC)样本数据库进行初步验证。

方法

构建稳定表达Cas9蛋白的人正常肺上皮BEAS-2B细胞系，在该细胞中感染含有77 441个单链向导RNA(sgRNA)、靶向19 114个人类基因的Brunello慢病毒文库，于裸鼠皮下移植。提取全瘤体细胞(Tumor)及移植前细胞(Input)的全基因组DNA，扩增sgRNA序列，建库进行深度测序。通过分析Tumor中sgRNA读数占比及Tumor与Input中sgRNA读数差异倍数的改变，筛选功能缺失后诱导BEAS-2B细胞致瘤性转化的抑癌基因，并利用公共数据平台进行初步验证。

结果

成功在稳定表达Cas9蛋白的BEAS-2B细胞中进行了文库感染及裸鼠皮下成瘤实验。只有感染文库的细胞在部分接种位点成瘤。测序结果显示Tumor中只检测到少数sgRNA

选取sgRNA的读数占总读数的比例

1%或有2个及以上sgRNA被显著富集的38个

基因为有效的人肺癌候选抑癌基因，包括

2、

PTEN

等已知抑癌基因及未在肺癌中报道过的候选抑癌基因，如

1、

PSENEN

等。富集分析发现候选抑癌基因在Notch和Hippo信号等参与癌症发生的关键生物学通路中富集。应用临床NSCLC样本数据库证实38个候选抑癌基因在NSCLC样本中均发生了突变，其中突变率＞2%的17个基因中有14个与

KRAS

或

53发生共突变，3个突变与预后相关；30个基因在NSCLC样本中的表达水平与正常癌旁组织有明显差异，17个基因的表达水平与患者的预后密切相关。

结论

利用体内全基因组CRISPR/Cas9技术筛选出38个人肺癌候选抑癌基因，并在临床NSCLC样本数据库中得到了初步验证。

Abstract

Objective

To identify new human lung cancer suppressors in a genome-wide screening library during neoplastic transformation of normal human lung epithelial cells using CRISPR/Cas9-mediated genome editing

and preliminarily validate the Results in non-small cell lung cancer (NSCLC) sample databases.

Methods

Human normal lung epithelial BEAS-2B cell line with stable expression of Cas9 protein was constructed

and then infected with the Brunello lentiviral library encompassing 77 441 single-guide RNAs (sgRNAs) targeting 19 114 genes. Then the obtained cells were transplanted subcutaneously into nude mice. Genomic DNA of tumor cells (Tumor) and pre-transplantation cells (Input) were extracted. The extracted DNA and plasmid library were amplified for deep sequencing. By analyzing the proportion of sgRNA reads in Tumor cells compared to the Input cells and the Log Fold Change (LFC) of sgRNA reads

tumor suppressor genes that can induce oncogenic transformation of BEAS-2B cells after knockout were screened

and preliminary validation were performed using public data platforms.

Results

Library infections in BEAS-2B cells stably expressing Cas9 protein and subcutaneous tumorigenesis in nude mice were successfully established. Only cells infected with the library formed tumors at the inoculation sites. Sequencing Results showed that only a small part of sgRNAs were detected in tumor cells. Here

genes with sgRNA reads accounting for more than 1% of the total reads or with 2 or more significantly enriched sgRNA were selected as effective candidate human lung tumor suppressor genes

including known tumor suppressor genes such as NF2 and PTEN

etc

and some candidate tumor suppressor genes (AP2M1 and PSENEN) had not been previously reported in lung cancer. Enrichment analysis revealed that these candidate tumor suppressor genes were enriched in key biological pathways involved in cancer

such as Notch and Hippo signaling pathways. It is confirmed in the clinical NSCLC sample databases that 38 candidate tumor suppressor genes were mutated in NSCLC samples. Among the 17 genes with mutation rates greater than 2%

14 genes were co-mutated with KRAS or TP53

and 3 ones were associated with the prognosis of NSCLC patients. There were 30 candidate genes with their expression in the samples of NSCLC different from those in para-tumor normal tissues

and the expression of 17 candidate genes were correlated with the prognosis of NSCLC patients.

Conclusion

By using an

in vivo

genome-wide CRISPR/Cas9 screening library

38 human lung cancer candidate suppressor genes are identified and preliminarily verified in the clinical NSCLC sample databases.

关键词

Keywords

references

SUNG H, FERLAY J, SIEGEL R L, et al . Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries[J ] . CA Cancer J Clin, 2021, 71(3): 209-249. DOI: 10.3322/caac.21660.

CHEN W Q, ZHENG R S, BAADE P D, et al . Cancer statistics in China, 2015[J ] . CA Cancer J Clin, 2016, 66(2): 115-132. DOI: 10.3322/caac.21338.

SMITH R A, ANDREWS K S, BROOKS D, et al . Cancer screening in the United States, 2019: a review of current American Cancer Society guidelines and current issues in cancer screening[J ] . CA Cancer J Clin, 2019, 69(3): 184-210. DOI: 10.3322/caac.21557.

CICEK Y, KOSAR P, ÖZTVRK Ö. Molecular genetics of lungcancer[J]. Eurasian J Pulmonol, 2018, 20(3): 111-117. DOI: 10.4103/ejop.ejop_33_18.

CHIN L, HAHN W C, GETZ G, et al . Making sense of cancer genomic data[J ] . Genes Dev, 2011, 25(6): 534-555. DOI: 10.1101/gad.2017311.

LI Y Y, KANG K, KRAHN J M, et al . A comprehensive genomic pan-cancer classification using The Cancer Genome Atlas gene expression data[J ] . BMC Genomics, 2017, 18(1): 508. DOI: 10.1186/s12864-017-3906-0.

PON J R, MARRA M A. Driver and passenger mutations in cancer[J]. Annu Rev Pathol, 2015, 10: 25-50. DOI: 10.1146/annurev-pathol-012414-040312.

CHEN S D, SANJANA N E, ZHENG K J, et al . Genome-wide CRISPR screen in a mouse model of tumor growth and metastasis[J ] . Cell, 2015, 160(6): 1246-1260. DOI: 10.1016/j.cell.2015.02.038.

SONG C Q, LI Y X, MOU H W, et al . Genome-wide CRISPR screen identifies regulators of mitogen-activated protein kinase as suppressors of liver tumors in mice[J ] . Gastroenterology, 2017, 152(5): 1161-1173. e1. DOI: 10.1053/j.gastro.2016.12.002.

REDDEL R R, KE Y, GERWIN B I, et al . Transformation of human bronchial epithelial cells by infection with SV40 or adenovirus-12 SV40 hybri d virus, or transfection via strontium phosphate coprecipitation with a plasmid containing SV40 early region genes[J ] . Cancer Res, 1988, 48(7): 1904-1909.

SANSON K R, HANNA R E, HEGDE M, et al . Optimized libraries for CRISPR-Cas9 genetic screens with multiple modalities[J ] . Nat Commun, 2018, 9(1): 5416. DOI: 10.1038/s41467-018-07901-8.

DOUDNA J A, CHARPENTIER E. Genome editing. The new frontier of genome engineering with CRISPR-Cas9[J]. Science, 2014, 346(6213): 1258096. DOI: 10.1126/science.1258096.

ZHOU Y Y, ZHOU B, PACHE L, et al . Metascape provides a biologist-oriented resource for the analysis of systems-level datasets[J ] . Nat Commun, 2019, 10(1): 1523. DOI: 10.1038/s41467-019-09234-6.

GAO J J, AKSOY B A, DOGRUSOZ U, et al . Integrative analysis of complex cancer genomics and clinical profiles using the cBioPortal[J ] . Sci Signal, 2013, 6(269): pl1. DOI: 10.1126/scisignal.2004088.

GREENMAN C, STEPHENS P, SMITH R, et al . Patterns of somatic mutation in human cancer genomes[J ] . Nature, 2007, 446(7132): 153-158. DOI: 10.1038/nature05610.

CUI Q H, MA Y, JARAMILLO M, et al . A map of human cancer signaling[J ] . Mol Syst Biol, 2007, 3: 152. DOI: 10.1038/msb4100200.

DING L, GETZ G, WHEELER D A, et al . Somatic mutations affect key pathways in lung adenocarcinoma[J ] . Nature, 2008, 455(7216): 1069-1075. DOI: 10.1038/nature07423.

NETWORK C G A R. Comprehensive genomic characterization of squamous cell lung cancers[J]. Nature, 2012, 489(7417): 519-525. DOI: 10.1038/nature11404.

NETWORK C G A R. Comprehensive molecular profiling of lung adenocarcinoma[J]. Nature, 2014, 511(7511): 543-550. DOI: 10.1038/nature13385.

Correction: LCE: an open web portal to explore gene expression and clinical associations in lung cancer[J]. Oncogene, 2020, 39(3): 718-719. DOI: 10.1038/s41388-019-1000-6.

AMSTAD P, REDDEL R R, PFEIFER A, et al . Neoplastic transformation of a human bronchial epithelial cell line by a recombinant retrovirus encoding vira l Harvey ras[J ] . Mol Carcinog, 1988, 1(3): 151-160. DOI: 10.1002/mc.2940010303.

秦爱平. 人支气管上皮细胞在致癌机制研究中的应用[J]. 卫生毒理学杂志, 2003, 17(3): 188-191. DOI: 10.16421/j.cnki.1002-3127.2003.03.020.

QIN A P. Application of human bronchial epithelial cells in the study of carcinogenesis mechanism[J]. J Heal Toxicol, 2003, 17(3): 188-191. DOI: 10.16421/j.cnki.1002-3127.2003.03.020.

KNUDSON A G JR. Mutation and cancer: statistical study of retinoblastoma[J]. Proc Natl Acad Sci USA, 1971, 68(4): 820-823. DOI: 10.1073/pnas.68.4.820.

BALMAIN A, GRAY J, PONDER B. The genetics and genomics ofcancer[J]. Nat Genet, 2003, 33(S3): 238-244. DOI: 10.1038/ng1107.

EVERS B, JASTRZEBSKI K, HEIJMANS J P M, et al . CRISPR knockout screening outperforms shRNA and CRISPRi in identifying essential genes[J ] . Nat Biotechnol, 2016, 34(6): 631-633. DOI: 10.1038/nbt.3536.

CHEN Z, FILLMORE C M, HAMMERMAN P S, et al . Non-small-cell lung cancers: a heterogeneous set of diseases[J ] . Nat Rev Cancer, 2014, 14(8): 535-546. DOI: 10.1038/nrc3775.

PETRILLI A M, FERNÁNDEZ-VALLE C. Role of merlin/NF2 inactivation in tumor biology[J]. Oncogene, 2016, 35(5): 537-548. DOI: 10.1038/onc.2015.125.

SONG X L, LI M L, WU W G, et al . Regulation of BMP2K in AP2M1-mediated EGFR internalization during the development of gallbladder cancer[J ] . Signal Transduct Target Ther, 2020, 5(1): 154. DOI: 10.1038/s41392-020-00250-3.

WILLIAMSON L, SAPONARO M, BOEING S, et al . UV irradiation induces a non-coding RNA that functionally opposes the protein encoded by the same gene[J ] . Cell, 2017, 168(5): 843-855.e13. DOI: 10.1016/j.cell.2017.01.019.

DUFFY M J, SYNNOTT N C, CROWN J. Mutant p53 as a target for cancer treatment[J]. Eur J Cancer, 2017, 83: 258-265. DOI: 10.1016/j.ejca.2017.06.023.

NAGY Á, LÁNCZKY A, MENYHÁRT O, et al . Validation of miRNA prognostic power in hepatocellular carcinoma using expression data of independent datasets[J ] . Sci Rep, 2018, 8(1): 9227. DOI: 10.1038/s41598-018-27521-y.

GÓMEZ-GIL V. Therapeutic implications of TGFβ in cancer treatment: a systematic review[J]. Cancers, 2021, 13(3): 379. DOI: 10.3390/cancers13030379.

李欢欢, 黄承浩. 基于CRISPR-Cas9的功能基因筛选研究进展[J]. 生物工程学报, 2018, 34(4): 461-472. DOI: 10.13345/j.cjb.170348.

LI H H, HUANG C H. Functional genetic screening using CRISPR-Cas9 system[J]. Chin J Biotechnol, 2018, 34(4): 461-472. DOI:10.13345/j.cjb.170348.

XU H, XIAO T F, CHEN C H, et al . Sequence determinants of improved CRISPR sgRNA design[J ] . Genome Res, 2015, 25(8): 1147-1157. DOI: 10.1101/gr.191452.115.

GILBERT L A, HORLBECK M A, ADAMSON B, et al . Genome-scale CRISPR-mediated control of gene repression and activation[J ] . Cell, 2014, 159(3): 647-661. DOI: 10.1016/j.cell.2014.09.029.

浏览量

下载量

CSCD

文章被引用时，请邮件提醒。

提交

工具集

关联资源

IKBKE在烟气凝集物诱导人支气管上皮细胞恶性转化过程中的作用

人Waardenburg综合征点突变SOX10 p.R106W小鼠模型的构建及听力表型初步研究

A549细胞中DBNLS232磷酸化调控丝状伪足形成及细胞迁移运动

敲除MT3R/Nqo2基因对大鼠睡眠/觉醒行为和EEG能谱的影响