[1]叶洲杰,王心睿.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[J].福建医药杂志,2023,45(04):101-106.
　YE Zhoujie,WANG Xinrui.FHL1 gene knockout and transcriptome analysis with CRISPR/Cas9 in human thyroid papillary carcinoma B-CPAP cells[J].FUJIAN MEDICAL JOURNAL,2023,45(04):101-106.
点击复制

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()
分享到：

《福建医药杂志》[ISSN:1002-2600/CN:35-1071/R]

卷:
45
期数:
2023年04期
页码:
101-106
栏目:
基础研究
出版日期:
2023-08-15

文章信息/Info

Title:
FHL1 gene knockout and transcriptome analysis with CRISPR/Cas9 in human thyroid papillary carcinoma B-CPAP cells
文章编号:
1002-2600(2023)04-0101-06
作者:
叶洲杰; 王心睿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)
Author(s):
YE Zhoujie;  WANG Xinrui
Fujian Provincial Hospital of Obstetrics and Gynecology,Medical Research Center, Fujian Maternity and Child Health Hospital, Fuzhou, Fujian 350001, China
关键词:
甲状腺乳头状癌;  FHL1;  RNA-Seq;  CRISPR/Cas9
Keywords:
papillary thyroid carcinoma;  FHL1;  RNA-Seq;  CRISPR/Cas9
分类号:
R736.1
文献标志码:
A
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-CPAPFHL1-/-细胞活率,细胞划痕实验计算B-CPAPFHL1-/-细胞迁移率,抽提B-CPAP细胞和B-CPAPFHL1-/-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-CPAPFHL1-/-细胞与B-CPAP细胞相比增殖速率、迁移能力加快。B-CPAP与B-CPAPFHL1-/-细胞转录组高通量测序分析结果表明,与B-CPAP细胞相比,B-CPAPFHL1-/-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-CPAPFHL1-/-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。
Abstract:
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-CPAPFHL1-/- cell viability was determined by CCK-8 assay,and B-CPAPFHL1-/-cell mobility was calculated by cell scratch assay. B-CPAP cells and B-CPAPFHL1-/- 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-CPAPFHL1-/- cells had faster proliferation and migration than B-CPAP cells. High-throughput sequencing analysis of B-CPAP and B-CPAPFHL1-/- cell transcriptome showed that 1 813 differential genes were detected in B-CPAPFHL1-/- 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-CPAPFHL1-/- 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.

参考文献/References:

[1] Nabhan F,Dedhia P H,Ringel M D.Thyroid cancer,recent advances in diagnosis and therapy[J].Int J Cancer,2021,149(5):984-992.
[2] Bergdorf K,Ferguson D C,Mehrad M,et al.Papillary thyroid carcinoma behavior:clues in the tumor microenvironment[J].Endocr Relat Cancer,2019,26(6):601-614.
[3] Luo H,Xia X Y,Kim G D,et al.Characterizing dedifferentiation of thyroid cancer by integrated analysis[J].Sci Adv,2021,7(31):eabf3657.
[4] Liu Y,Wang C,Cheng P,et al.FHL1 Inhibits the progression of colorectal cancer by regulating the Wnt/β-Catenin signaling pathway[J].J Cancer,2021,12(17):5345-5354.
[5] Hartog L,Zhao J,Reynolds J,et al.Factors influencing the severity and progression of respiratory muscle dysfunction in myotonic dystrophy type 1[J].Front Neurol,2021(12):658532.
[6] Bolger A M,Lohse M,Usadel B.Trimmomatic:a flexible trimmer for Illumina sequence data[J].Bioinformatics,2014,30(15):2114-2120.
[7] Kim D,Langmead B,Salzberg S L.HISAT:a fast spliced aligner with low memory requirements[J].Nat Methods,2015,12(4):357-360.
[8] Kanehisa M,Araki M,Goto S,et al.KEGG for linking genomes to life and the environment[J].Nucleic Acids Res,2008,36(Database issue):D480-D484.
[9] Gilson P,Drouot G,Witz A,et al.Emerging Roles of DDB2 in Cancer[J].Int J Mol Sci,2019,20(20):5168.
[10] Zhang H,Wang Y,Ding H.COL4A1,negatively regulated by XPD and miR-29a-3p,promotes cell proliferation,migration,invasion and epithelial-mesenchymal transition in liver cancer cells[J].Clin Transl Oncol,2021,23(10):2078-2089.
[11] Wang D S,Zhao C L,Gao L L,et al.NPNT promotes early-stage bone metastases in breast cancer by regulation of the osteogenic niche[J].J Bone Oncol,2018(13):91-96.

相似文献/References:

[1]陈琼,叶小芬,王康健,等.伴有不同甲状腺疾病的乳头状癌颈中央区淋巴结转移预测模型建立与验[J].福建医药杂志,2023,45(06):4.
　CHEN Qiong,YE Xiaofen,WANG Kangjian,et al.Prediction of papillary thyroid metastases to the central compartment: proposal of a model taking into consideration other thyroid conditions[J].FUJIAN MEDICAL JOURNAL,2023,45(04):4.

备注/Memo

备注/Memo:
基金项目:福建省卫生健康青年科研课题项目(2019-1-14)
1 通信作者,E-mail:wanxiru@sjtu.edu.cn
更新日期/Last Update: 2023-08-15