构建EIF5A2基因敲除Cal27细胞株及功能分析
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摘要
目的运用CRISPR/Cas9技术构建EIF5A2基因敲除Cal27细胞系,观察EIF5A2在口腔鳞状细胞癌增殖与迁移中的作用。方法根据EIF5A2基因结构域,设计2个CRISPR靶向位点,利用pGL3-U6-sgRNA-PGK-puromycin构建2个导向RNA(sgRNA)质粒。将sgRNA质粒与pST1374-NLS-flag-linker-Cas9共转染至Cal27细胞中,用嘌呤霉素和杀稻瘟菌素筛选,并挑取单克隆。将单克隆细胞的DNA进行目的片段扩增,经凝胶电泳及测序鉴定后筛选出大片段敲除细胞株Cal27-2C5及碱基突变株Cal27-2D1。real time PCR及WesternBlot鉴定两株细胞系中EIF5A2的表达情况。用Transwell实验及CCK8分别检测EIF5A2敲除对Cal27细胞的体外迁移能力及体外增殖能力的影响。结果与对照组相比,Cal27-2C5及Cal27-2D1细胞株中EIF5A2在mRNA水平上表达均明显降低。在蛋白水平上,Cal27-2C5及Cal27-2D1细胞株中EIF5A2蛋白均基本不表达。敲除EIF5A2可明显降低Cal27细胞的体外迁移能力,而对Cal27细胞体外增殖能力没有明显影响。结论成功构建EIF5A2基因敲除Cal27细胞株Cal27-2C5和Cal27-2D1,并初步验证EIF5A2可以影响Cal27细胞的体外迁移能力。
Objective To construct EIF5 A2 knock-out Cal27 cell line by CRISPR/Cas9 technology, and to observe the role of EIF5 A2 in the proliferation and migration of oral squamous cell carcinoma. Methods Two CRISPR targeting sites were designed based on the EIF5 A2 gene domains. Two single guide RNA(sgRNA) plasmids were constructed using pGL3-U6-sgRNA-PGK-puromycin. The sgRNA plasmids were transfected into Cal27 cells with plasmid pST1374-NLS-flag-linker-Cas9, and the cells were selected with puromycin and blasticidin. Then monoclonal strains were picked. The target DNA of the monoclonal cells was amplified by PCR and identified by gel electrophoresis and sequencing. The large fragment knock-out cell line Cal27-2 C5 and the base mutant Cal27-2 D1 were screened out. Real-time PCR and Western Blot were used to identify the expression of EIF5 A2 in two cell lines. Finally, Transwell assay and CCK8 were used to examine the effect of EIF5 A2 knockout on in vitro migration ability and in vitro proliferation of Cal27 cells. Results Compared with the control group, the expression of EIF5 A2 in Cal27-2 C5 and Cal27-2 D1 cell lines was significantly up-regulated at mRNA level. EIF5 A2 was almost not expressed in both Cal27-2 C5 and Cal27-2 D1 cell lines at protein levels. Knockout EIF5 A2 significantly reduced the in vitro migration of Cal27 cells, but had no significant effect on the proliferation of Cal27 cells in vitro. Conclusion The EIF5 A2 knock out cell line Cal27-2 C5 and Cal27-2 D1 was constructed and EIF5 A2 could affect the migration ability of Cal27 cells in vitro.
Objective To construct EIF5 A2 knock-out Cal27 cell line by CRISPR/Cas9 technology, and to observe the role of EIF5 A2 in the proliferation and migration of oral squamous cell carcinoma. Methods Two CRISPR targeting sites were designed based on the EIF5 A2 gene domains. Two single guide RNA(sgRNA) plasmids were constructed using pGL3-U6-sgRNA-PGK-puromycin. The sgRNA plasmids were transfected into Cal27 cells with plasmid pST1374-NLS-flag-linker-Cas9, and the cells were selected with puromycin and blasticidin. Then monoclonal strains were picked. The target DNA of the monoclonal cells was amplified by PCR and identified by gel electrophoresis and sequencing. The large fragment knock-out cell line Cal27-2 C5 and the base mutant Cal27-2 D1 were screened out. Real-time PCR and Western Blot were used to identify the expression of EIF5 A2 in two cell lines. Finally, Transwell assay and CCK8 were used to examine the effect of EIF5 A2 knockout on in vitro migration ability and in vitro proliferation of Cal27 cells. Results Compared with the control group, the expression of EIF5 A2 in Cal27-2 C5 and Cal27-2 D1 cell lines was significantly up-regulated at mRNA level. EIF5 A2 was almost not expressed in both Cal27-2 C5 and Cal27-2 D1 cell lines at protein levels. Knockout EIF5 A2 significantly reduced the in vitro migration of Cal27 cells, but had no significant effect on the proliferation of Cal27 cells in vitro. Conclusion The EIF5 A2 knock out cell line Cal27-2 C5 and Cal27-2 D1 was constructed and EIF5 A2 could affect the migration ability of Cal27 cells in vitro.
引文
1刘彤华.诊断病理学.3版.北京:人民卫生出版社,2013.13.
2 Woolgar JA,Rogers SN,Lowe D,et al.Cervical lymph node metastasis in oral cancer:the importance of even microscopic extracapsular spread.Oral Oncology,2003,39(2):130-136.
3 Miyahara M,Tanuma J,Sugihara K,et al.Tumor lymphangiogenesis correlates with lymph node metastasis and clinicopathologic parameters in oral squamous cell carcinoma.Cancer,2007,110(6):1287-1294.
4付洁,刘曼,苏颖,等.DNA甲基化和组蛋白乙酰化对人口腔鳞癌细胞中KLF4基因的调控研究.北京口腔医学,2016,24(10):241-245.
5 Guan XY,Sham JS,Tang TC,et al.Isolation of a novel candidate oncogene within a frequently amplified region at 3q26 in ovarian cancer.Cancer Res,2001,61(9):3806-3809.
6 Li Y,Fu L,Li JB,et al.Increased expression of EIF5A2,via hypoxia or gene amplification,contributes to metastasis and angiogenesis of esophageal squamous cell carcinoma.Gastroenterology,2014,146(7):1701-1713.e9.
7 Jenkins ZA,Haag PG,Johansson HE.Human e IF5A2 on chromosome 3q25-q27 is a phylogenetically conserved vertebrate variant of eukaryotic translation initiation factor 5A with tissuespecific expression.Genomics,2001,71(1):101-109.
8 Tsang FH,Au V,Lu WJ,et al.Prognostic marker microRNA-125b inhibits tumorigenic properties of hepatocellular carcinoma cells via suppressing tumorigenic molecule e IF5A2.Dig Dis Sci,2014,59(10):2477-2487.
9 Liu T,Shen JK,Li Z,et al.Development and potential applications of CRISPR-Cas9 genome editing technology in sarcoma.Cancer Lett,2016,373(1):109-118.
10 Paquet D,Kwart D,Chen A,et al.Efficient introduction of specific homozygous and heterozygous mutations using CRISPR/Cas9.Nature,2016,533(7601):125-129.
11 Canver MC,Bauer DE,Dass A,et al.Characterization of genomic deletion efficiency mediated by clustered regularly interspaced short palindromic repeats(CRISPR)/Cas9 nuclease system in mammalian cells.J Biol Chem,2014,289(31):21312-21324.
2 Woolgar JA,Rogers SN,Lowe D,et al.Cervical lymph node metastasis in oral cancer:the importance of even microscopic extracapsular spread.Oral Oncology,2003,39(2):130-136.
3 Miyahara M,Tanuma J,Sugihara K,et al.Tumor lymphangiogenesis correlates with lymph node metastasis and clinicopathologic parameters in oral squamous cell carcinoma.Cancer,2007,110(6):1287-1294.
4付洁,刘曼,苏颖,等.DNA甲基化和组蛋白乙酰化对人口腔鳞癌细胞中KLF4基因的调控研究.北京口腔医学,2016,24(10):241-245.
5 Guan XY,Sham JS,Tang TC,et al.Isolation of a novel candidate oncogene within a frequently amplified region at 3q26 in ovarian cancer.Cancer Res,2001,61(9):3806-3809.
6 Li Y,Fu L,Li JB,et al.Increased expression of EIF5A2,via hypoxia or gene amplification,contributes to metastasis and angiogenesis of esophageal squamous cell carcinoma.Gastroenterology,2014,146(7):1701-1713.e9.
7 Jenkins ZA,Haag PG,Johansson HE.Human e IF5A2 on chromosome 3q25-q27 is a phylogenetically conserved vertebrate variant of eukaryotic translation initiation factor 5A with tissuespecific expression.Genomics,2001,71(1):101-109.
8 Tsang FH,Au V,Lu WJ,et al.Prognostic marker microRNA-125b inhibits tumorigenic properties of hepatocellular carcinoma cells via suppressing tumorigenic molecule e IF5A2.Dig Dis Sci,2014,59(10):2477-2487.
9 Liu T,Shen JK,Li Z,et al.Development and potential applications of CRISPR-Cas9 genome editing technology in sarcoma.Cancer Lett,2016,373(1):109-118.
10 Paquet D,Kwart D,Chen A,et al.Efficient introduction of specific homozygous and heterozygous mutations using CRISPR/Cas9.Nature,2016,533(7601):125-129.
11 Canver MC,Bauer DE,Dass A,et al.Characterization of genomic deletion efficiency mediated by clustered regularly interspaced short palindromic repeats(CRISPR)/Cas9 nuclease system in mammalian cells.J Biol Chem,2014,289(31):21312-21324.
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