应用CRISPR/Cas9技术敲除甲状腺乳头状癌B-CPAP细胞FHL1基因及转录组分析  

FHL1 gene knockout and transcriptome analysis with CRISPR/Cas9 in human thyroid papillary carcinoma B-CPAP cells

作　　者：叶洲杰 王心睿 YE Zhoujie;WANG Xinrui(Fujian Provincial Hospital of Obstetrics and Gynecology,Medical Research Center,Fujian Maternity and Child Health Hospital,Fuzhou,Fujian 350001,China)

机构地区：[1]福建省妇产医院,福建省妇幼保健院医学研究中心,福州350001

出　　处：《福建医药杂志》2023年第4期101-106,共6页Fujian Medical Journal

基　　金：福建省卫生健康青年科研课题项目(2019-1-14)。

摘　　要：目的 探究FHL1在甲状腺瘤细胞中参与的细胞生物学功能,为后续研究FHL1基因介导甲状腺癌发生发展的分子机制提供依据。方法 运用CRISPR/Cas9基因编辑技术在甲状腺乳头状癌B-CPAP细胞系中大片段敲除FHL1基因,CCK-8实验测定B-CPAP^(FHL1-/-)细胞活率,细胞划痕实验计算B-CPAP^(FHL1-/-)细胞迁移率,抽提B-CPAP细胞和B-CPAP^(FHL1-/-)细胞RNA用于转录组高通量测序,并进行表达差异基因KEGG与GO富集分析。结果 实验结果显示B-CPAP细胞中FHL1基因缺失35 995 bp,导致蛋白功能被破坏。B-CPAP^(FHL1-/-)细胞与B-CPAP细胞相比增殖速率、迁移能力加快。B-CPAP与B-CPAP^(FHL1-/-)细胞转录组高通量测序分析结果表明,与B-CPAP细胞相比,B-CPAP^(FHL1-/-)细胞蛋白编码基因表达量分析检测到1 813个差异基因,其中1 125个基因上调,688个基因下调。KEGG通路富集分析发现细胞外基质受体交互作用、肿瘤信号通路、细胞黏附分子信号通路基因表达明显较高,这3条信号通路对肿瘤信号转导、侵袭及转移具有重要作用。对差异表达基因进行GO富集分析,发现上述3条信号通路的多种上调和下调表达基因。结论 B-CPAP^(FHL1-/-)细胞可能通过胞外基质受体交互作用、肿瘤信号通路、细胞黏附分子等信号通路上调相关基因表达,从而提升细胞增殖、迁移能力。Objective To explore the cellular biological function of FHL1 in thyroid tumor cells,and to provide a basis for further research on the molecular mechanism of FHL1 gene mediating the occurrence and development of thyroid cancer.Methods Using CRISPR/Cas9 gene editing technology,FHL1 gene was knocked out in large fragments of thyroid papillary carcinoma B-CPAP cell line,B-CPAP^(FHL1-/-) cell viability was determined by CCK-8 assay,and B-CPAP^(FHL1-/-)cell mobility was calculated by cell scratch assay.B-CPAP cells and B-CPAP^(FHL1-/-) cell RNA were extracted for high-throughput transcriptome sequencing and enrichment analysis of expression differential genes KEGG and GO.Results The results showed a 35 995 bp deletion of the FHL1 gene in B-CPAP cells,resulting in a breakdown of the protein's function.B-CPAP^(FHL1-/-) cells had faster proliferation and migration than B-CPAP cells.High-throughput sequencing analysis of B-CPAP and B-CPAP^(FHL1-/-) cell transcriptome showed that 1 813 differential genes were detected in B-CPAP^(FHL1-/-) cell transcriptome compared with B-CPAP cells,among which 1 125 genes were up-regulated and 688 genes were down-regulated.KEGG pathway enrichment analysis showed that the gene expressions of extracellular matrix receptor interaction,tumor signaling and cell adhesion molecule signaling pathways were significantly higher,and the three signaling pathways played an important role in tumor signal transduction,invasion and metastasis.GO enrichment analysis of differentially expressed genes showed that a variety of up-regulated and down-regulated genes were found in the above three signaling pathways.Conclusion B-CPAP^(FHL1-/-) cells may up-regulate the expression of related genes through signaling pathways such as extracellular matrix receptor interaction,tumor signaling,and cell adhesion molecules,thereby enhancing cell proliferation and migration.

关 键 词：甲状腺乳头状癌 FHL1 RNA-SEQ CRISPR/Cas9 

分 类 号：R736.1[医药卫生—肿瘤]