基于CRISPR/Cas9技术构建CELF6基因敲除细胞系并探讨CELF6与p53基因的关系

目的:构建CELF6基因敲除细胞系并探讨CELF6与p53基因之间的关系。方法:CRISPR/Cas9双载体慢病毒系统通过两个慢病毒载体分别向细胞中导入Cas9蛋白和sg RNA序列表达框,从而实现对CELF6基因的敲除。通过Surveyor(错配酶法)检测sg RNA活性并利用Western blot(蛋白质免疫印迹)检测CELF6的敲除效率。进一步利用CCK-8试剂盒检测敲除CELF6和过表达CELF6对细胞增殖的影响。利用公开可用的癌症基因组图谱(TCGA)数据库分析CELF6在多种肿瘤组织中的表达情况。结果:CELF6基因敲除的HCT116细胞系成功构建。Western blot检测发现CELF6基因敲除的细胞系中p53、p-RB蛋白的表达水平显著下调,而CELF6过表达的细胞中p53、p-RB蛋白的表达水平明显上调。CCK-8实验结果显示敲除CELF6基因后,细胞增殖活性显著增强;而过表达CELF6后,细胞的增殖活性受到明显抑制。生物信息学分析发现CELF6在结肠癌、胶质母细胞瘤、子宫内膜癌、肾嫌色细胞癌、乳腺癌、甲状腺癌等肿瘤组织中显著低表达。结论:推测CELF6是一种新的潜在肿瘤抑制基因,其可能在肿瘤的发生、发展过程中发挥重要作用。

Construction of CELF6 Knockout Cell Line Based on CRISPR / Cas9 Technique and Investigation of the Relationship between CELF6 and p53 Gene

ABSTRACT Objective: Construction of CELF6 knockout cell line and investigation of the relationship between CELF6 and p53 genes. Methods: The CRISPR/Cas9 dual vector lentiviral system introduces the Cas9 protein and sgRNA sequence expression box into cells through two lentiviral vectors, thereby knocking out the CELF6 gene. The sgRNA activity was detected by Surveyor method and the knockout efficiency of CELF6 was detected by Western blot. The effect of knockout CELF6 and overexpression of CELF6 on cell proliferation was further examined using the CCK-8 kit. The expression of CELF6 in various tumor tissues was analyzed using the publicly available Cancer Genome Atlas (TCGA) database. Results: The CELF6 knockout HCT116 cell line was successfully constructed. Western blot analysis showed that the expression levels of p53 and p-RB proteins in CELF6 knockout cell line was significantly down-regulated, while the expression levels of p53 and p-RB proteins in CELF6 overexpressing cell was significantly up-regulated. The results of CCK-8 showed that the cell proliferation activity was significantly enhanced in CELF6-KO cell lines, while in CELF6 overexpression cells, cell proliferation was significantly inhibited. Bioinformatics analysis found that CELF6 was significantly down-regulated in colon adenocarcinoma, glioblastoma multiforme, uterine corpus endometrial carcinoma, chromophobe cell carcinoma, breast invasive carcinoma, thyroid carcinoma and other tumor tissues. Conclusion: It is speculated that CELF6 is a new potential tumor suppressor gene, which may play an important role in the occurrence and development of tumors.