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ARID2基因敲除对肝癌细胞系Hep3B增殖及基因表达的影响
作者:周彦池 赵宏 
单位:100021 北京 国家癌症中心/国家肿瘤临床医学研究中心/中国医学科学院北京协和医学院肿瘤医院(周彦池、赵宏) 
关键词:肝肿瘤 基因 ARID2 细胞系 Hep3B CRISPR/Cas9技术 生物信息学分析 
分类号:R735.7
出版年,卷(期):页码:2019,44(6):451-458
摘要:

 [摘要]  目的  探讨ARID2敲除对细胞增殖的影响,以及敲除细胞与野生型Hep3B细胞间基因表达的差异。方法  构建ARID2敲除的lentiCRISPRv2质粒,转染Hep3B细胞,嘌呤霉素筛选后通过流式细胞仪分选单细胞,并培养获得单克隆细胞株;通过Western blottingSanger测序鉴定ARID2敲除细胞株;通过CCK-8实验检测ARID2敲除对Hep3B细胞增殖的影响;通过RNA-seq分析差异表达基因,并通过实时定量PCR(RT-qPCR)技术进行验证。通过京都基因与基因组百科全书数据库通路注释(KEGG Pathway)、基因本体生物学过程(GO Biological Processes)富集分析以及基因集富集分析(GSEA) ARID2可能参与的生物学功能。结果  成功构建两株ARID2敲除的Hep3B人肝癌细胞系。与野生型Hep3B 细胞相比,ARID2敲除细胞增殖明显加快(P<0.05)RNA-seq分析共检测到85个差异表达基因,其中17个基因上调, 68个基因下调。通过RT-qPCR对其中10个差异表达基因进行验证,验证结果与RNA-seq结果一致。KEGG PathwayGO Biological Processes富集分析以及GSEA结果表明,ARID2基因与蛋白质的加工及转运、趋化因子信号通路、Wnt信号通路、补体与凝血级联反应、上皮间质转化(EMT)、糖酵解、TGF-β信号通路、TNF-α/NF-κB信号通路等生物学过程相关。结论  ARID2敲除可以促进Hep3B细胞系的增殖;ARID2可能通过多种生物学过程,对肿瘤细胞增殖、侵袭、迁移,以及肿瘤微环境产生影响。

 [Abstract]  Objective  To construct ARID2 knockout human liver cancer cell line Hep3B by CRISPR/Cas9 system, explore the effect of ARID2 knockout on the proliferation of Hep3B, and the differences in gene expression between wild type and ARID2 knockout Hep3B cell lines. Methods  The plasmid lentiCRISPRv2 was constructed with ARID2 knockout plasmid, and then transfected Hep3B cells; The positive cells were selected by puromycin, sorted by flow cytometry and cultured to obtain the monoclonal cell lines; ARID2 knockout Hep3B cell lines were identified by Western blotting and Sanger sequencing; The effect of ARID2 knockout on the proliferation of Hep3B cell line was detected by CCK-8 method; RNA-seq was used to analyze the differentially expressed genes between wild type Hep3B cells and ARID2 knockout Hep3B cells, and the results of RNA-seq were validated by Real-time quantitative PCR (RT-qPCR). The possible biology functions of ARID2 were explored through Kyoto Encyclopedia of Genes and Genomes Pathway (KEGG Pathway), GO Biological Processes and Gene set enrichment analysis (GSEA). Results  Two ARID2 knockout Hep3B cell lines were successfully constructed. Compared with the wild type cell line, the proliferation of ARID2 knockout cell lines was significantly accelerated (P<0.05). A total of 85 differentially expressed genes were identified by RNA-seq analysis between ARID2 knockout cell lines and wild type cell line, of which 17 genes were up-regulated and 68 down-regulated. The mRNA expression of 10 differentially expressed genes were validated by RT-qPCR, the verification results were consistent with that by RNA-seq. Results of KEGG Pathway, GO Biological Processes and GSEA indicated that ARID2 genes might be involved in such biological processes as protein processing and transport, chemokine signaling pathway, Wnt signaling pathway, complement and coagulation cascade reaction, epithelial-mesenchymal transition (EMT), glycolysis, TGF-β signaling pathway, and TNF-α/NF-κB signaling pathway, et al. Conclusion  ARID2 knockout can promote proliferation of Hep3B cell line; and ARID2 may play an important role by variety of biological processes in tumor proliferation, invasion, metastasis and tumor microenvironment.

基金项目:
国家自然科学基金(81201967,81672461);国家科技重大专项项目(2017ZX10201201-007-003);北京市自然科学基金(7132193);中国医学科学院医学与健康科技创新工程项目(2017-I2M-4-002)
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