应用CRISPR-Cas9技术构建NFIB敲除的膀胱癌细胞株及其转录组学分析  

Construction of NFIB Knock-Out Bladder Cancer Cell Line by CRISPR-Cas9 Technology and Its Transcriptomics Analysis

作　　者：沈鹭 孙越 邬燕倩 蒋莹 洪叶挺 吕建新 SHEN Lu;SUN Yue;WU Yanqian;JIANG Ying;HONG Yeting;LYU Jianxin(School of Laboratory Medicine and Life Science,Wenzhou Medical University,Wenzhou 325000,China;School of Laboratory Medicine and Bioengineering,Hangzhou Medical College,Hangzhou 311399,China)

机构地区：[1]温州医科大学检验医学院、生命科学学院,温州325000 [2]杭州医学院检验医学院、生物工程学院,杭州311399

出　　处：《中国细胞生物学学报》2022年第7期1248-1257,共10页Chinese Journal of Cell Biology

基　　金：领军创新团队-重大疾病分子机制和检验诊断医疗器械研发项目(批准号:CXLJ202101);国家自然科学基金(批准号:81801513);浙江省高校基本科研经费(批准号:KYZD202003)资助的课题。

摘　　要：为了探究NFIB(nuclear factor I/B)基因在膀胱癌中的生物学功能,该研究应用CRISPRCas9技术构建NFIB敲除的膀胱癌细胞株,然后通过转录组学技术分析对照细胞NFIB-NC与NFIB敲除细胞株NFIB-KO-I和NFIB-KO-II之间的差异表达基因,同时对其进行KEGG富集分析,并通过Western blot和qRT-PCR技术对转录组学结果进行验证。结果显示,与对照细胞NFIB-NC相比,NFIB敲除细胞株NFIB-KO-I中有134个差异表达基因,其中上调基因62个,下调基因72个;NFIB-KO-II中有131个差异表达基因,表达上调和下调的基因分别为50个和81个。KEGG分析结果显示,差异表达基因与PI3K-AKT信号通路密切相关,Western blot结果证实敲除NFIB基因后,膀胱癌细胞中的p-AKT水平显著上调。qRT-PCR结果表明,敲除NFIB后,ITGA4基因表达水平上调,TNC和ANGPT4基因表达水平下调。该研究初步揭示了NFIB基因在膀胱癌细胞中调控的分子信号通路,也为后续研究NFIB基因介导膀胱癌发生发展的分子机制提供了依据。In order to explore the biological function of NFIB gene in bladder cancer,CRISPR-Cas9 technology was utilized to knock out NFIB gene in bladder cancer cell line,and transcriptomics analysis was used to detect the DEGs(differentially expressed genes)between control cell NFIB-NC and NFIB knock out cells NFIBKO-I and NFIB-KO-II,and KEGG enrichment analysis was performed.The results of transcriptomics analysis were also verified by Western blot and qRT-PCR.Compared with the control cell NFIB-NC,there were 134 DEGs in NFIB knock-out cell line NFIB-KO-I,including 62 upregulated genes and 72 downregulated genes,and 131 DEGs in NFIB knock-out cell line NFIB-KO-II,including 50 upregulated genes and 81 downregulated genes.The results of KEGG enrichment analysis showed that DEGs were tightly related to PI3K-AKT signaling pathway,and the results of Western blot confirmed that the expression level of p-AKT was significantly upregulated in bladder cancer cell after knocking out NFIB.The results of qRT-PCR showed that after knocking out NFIB,the expression level of ITGA4 was upregulated,and the levels of TNC and ANGPT4 were downregulated.The study revealed the regulated molecular signaling pathways of NFIB,which could provide a basis for the follow-up research on the molecular mechanism of NFIB mediating the occurrence and development of bladder cancer.

关 键 词：NFIB基因 CRISPR-Cas9 膀胱癌 转录组学分析 差异表达基因 

分 类 号：R737.14[医药卫生—肿瘤]