敲除ARID1A对人肝癌细胞系PLC/PRF/5基因表达调控的影响

基础医学与临床 ›› 2020, Vol. 40 ›› Issue (4): 501-505.

敲除ARID1A对人肝癌细胞系PLC/PRF/5基因表达调控的影响

王町町, 梁俊波, 王晓月^*

中国医学科学院基础医学研究所北京协和医学院基础学院生物化学与分子生物学系, 北京 100005

收稿日期:2019-12-16 修回日期:2020-01-11 出版日期:2020-04-05 发布日期:2020-04-06
通讯作者: *pumcwangxy@163.com
基金资助:
中国医学科学院基本科研业务费(2015RC310002)

Effects of ARID1A loss on transcriptional regulation in hepatocellular carcinoma cell line PLC/PRF/5

WANG Ding-ding, LIANG Jun-bo, WANG Xiao-yue^*

Department of Biochemistry and Molecular Biology, Institute of Basic Medical Sciences CAMS, Istitute of Basic Medicine PUMC, Beijing 100005, China

Received:2019-12-16 Revised:2020-01-11 Online:2020-04-05 Published:2020-04-06
Contact: *pumcwangxy@163.com

摘要/Abstract

摘要： 目的探讨肝癌细胞系PLC/PRF/5中敲除ARID1A后对基因表达调控的影响。方法利用CRISPR-Cas9基因编辑技术敲除PLC/PRF/5细胞中ARID1A。然后分别对PLC/PRF/5 ARID1A野生型与敲除型细胞进行全转录组测序分析(RNA-seq),并用DESeq2 鉴定ARID1A敲除后差异表达基因。最后,使用Metascape数据库对差异表达基因进行GO功能富集分析。结果成功构建PLC/PRF/5 ARID1A敲除细胞系。利用RNA-seq共筛选出978个差异表达基因,包括480个表达上调差异基因和498个表达下调差异基因。GO功能富集分析显示差异表达基因主要集中在细胞黏附、激素水平调节、激素代谢和MAP激酶活性调节等生物学过程。结果 PLC/PRF/5肝癌细胞中,ARID1A可调控大量基因的表达,为进一步研究ARID1A在肝癌发生发展中的作用机制提供了新的线索。

关键词: ARID1A, CRISPR, 全转录组测序, 人肝癌细胞系PLC/PRF/5

Abstract: Objective To explore the transcriptional alterations regulated by ARID1A in hepatocellular carcinoma cell line PLC/PRF/5. Methods CRISPR/Cas9 technology was utilized to generate ARID1A knockout cells. RNA-seq was then performed in both ARID1A intact and ARID1A null PLC/PRF/5 cells to generate gene expression profiles. Differentially expressed genes were identified using DESeq2. Functional enrichment analysis of differentially expressed genes was performed using metascape database. Results Successful generation of the ARID1A knockout PLC/PRF/5 cells was confirmed by Western blot and Sanger sequencing. A total of 978 differentially expressed genes were identified by RNA-seq, among which the expression of 480 genes was up-regulated and that of 498 genes was down-regulated. These differentially expressed genes were mainly enriched in several biological processes including cell adhesion, regulation of hormone levels, hormone metabolism and regulation of MAP kinase activity. Conclusions The results indicate that ARID1A loss induces alterations in transcription in PLC/PRF/5, which may provide novo clues for elucidating the underlying mechanism of ARID1A in tumorigenesis.

Key words: ARID1A, CRISPR, RNA-seq, hepatocellular carcinoma cell line PLC/PRF/5

中图分类号:

王町町, 梁俊波, 王晓月. 敲除ARID1A对人肝癌细胞系PLC/PRF/5基因表达调控的影响[J]. 基础医学与临床, 2020, 40(4): 501-505.

WANG Ding-ding, LIANG Jun-bo, WANG Xiao-yue. Effects of ARID1A loss on transcriptional regulation in hepatocellular carcinoma cell line PLC/PRF/5[J]. Basic & Clinical Medicine, 2020, 40(4): 501-505.

参考文献

[1]Wilson BG, Roberts CW. SWI/SNF nucleosome remod-ellers and cancer[J]. Nat Rev Cancer, 2011,11:481-492.
[2]Kadoch C, Crabtree GR. Mammalian SWI/SNF chromatin remodeling complexes and cancer: mechanistic insights gained from human genomics[J]. Sci Adv, 2015,1:e1500447. doi: 10.1126/sciadv.1500447.
[3]Kadoch C, Hargreaves DC, Hodges C, et al. Proteomic and bioinformatic analysis of mammalian SWI/SNF complexes identifies extensive roles in human malignancy[J]. Nat Genet, 2013,45:592-601.
[4]Mathur R, Alver BH, San Roman AK, et al. ARID1A loss impairs enhancer-mediated gene regulation and drives colon cancer in mice[J]. Nat Genet, 2017,49:296-302.
[5]Shen J, Peng Y, Wei L, et al. ARID1A deficiency impairs the DNA damage checkpoint and sensitizes cells to PARP inhibitors[J]. Cancer Discov, 2015,5:752-767.
[6]Guan B, Wang TL, Shih Ie M. ARID1A, a factor that promotes formation of SWI/SNF-mediated chromatin remodeling, is a tumor suppressor in gynecologic cancers[J]. Cancer Res, 2011,71:6718-6727.
[7]Suryo Rahmanto Y, Jung JG, Wu RC, et al. Inactivating ARID1A tumor suppressor enhances TERT transcription and maintains telomere length in cancer cells[J]. J Biol Chem, 2016,291:9690-9699.
[8]Pertea M, Kim D, Pertea GM, et al. Transcript-level expression analysis of RNA-seq experiments with HISAT, StringTie and Ballgown[J]. Nat Protoc, 2016,11:1650-1667.
[9]Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2[J]. Genome Biol, 2014,15:550. doi: 10.1186/s13059-014-0550-8.
[10]Zhou Y, Zhou B, Pache L, et al. Metascape provides a biologist-oriented resource for the analysis of systems-level datasets[J]. Nat Commun, 2019,10:1523. doi:10.1038/s41467-019-09234-6.
[11]Inoue H, Furukawa T, Giannakopoulos S, et al. Largest subunits of the human SWI/SNF chromatin-remodeling complex promote transcriptional activation by steroid hormone receptors[J]. J Biol Chem, 2002,277:41674-41685.
[12]Li C, Xu ZL, Zhao Z, et al. ARID1A gene knockdown promotes neuroblastoma migration and invasion[J]. Neoplasma, 2017,64:367-376.
[13]Kim EK, Choi EJ. Compromised MAPK signaling in human diseases: an update[J]. Arch Toxicol, 2015,89:867-882.