| 刘瑞琦,王 洁,雷 超,刘一睿,王雅婷,张竞方.利用CRISPR/Cas9技术靶向KRAS或TP53突变抑制SW620细胞的增殖[J].现代生物医学进展英文版,2023,(21):4001-4006. |
| 利用CRISPR/Cas9技术靶向KRAS或TP53突变抑制SW620细胞的增殖 |
| Targeting KRAS or TP53 Mutation by CRISPR/Cas9 Inhibited the Proliferation of SW620 Cells |
| Received:April 06, 2023 Revised:April 28, 2023 |
| DOI：10.13241/j.cnki.pmb.2023.21.001 |
| 中文关键词: CRISPR/Cas9 结直肠癌 KRAS TP53 |
| 英文关键词: CRISPR/Cas9 Colorectal cancer KRAS TP53 |
| 基金项目:国家自然科学青年基金项目（31701280） |
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| 中文摘要: |
| 摘要 目的：运用CRISPR/Cas9基因编辑工具研究靶向SW620细胞系中KRAS或TP53突变对细胞增殖活性的影响。方法：针对SW620细胞中KRAS和TP53的突变位点设计sgRNA，并利用TIDE法检测sgRNA的切割效率。通过细胞增殖实验检测靶向KRAS或TP53突变后SW620细胞增殖活性的改变，并应用Annexin V-FITC/PI双染法检测细胞凋亡水平的变化。结果：分别构建了靶向SW620细胞系中KRAS和TP53突变的sgRNA质粒，并通过TIDE分析验证了sgRNA的内源切割效率；细胞增殖实验及细胞凋亡检测显示，靶向突变的KRAS或TP53基因后，SW620细胞增殖活性明显减弱，凋亡水平明显升高（P<0.05）。结论：本研究基于CRISPR/Cas9技术实现了对SW620细胞系中突变的KRAS和TP53的基因编辑，发现靶向KRAS或TP53突变能够明显抑制SW620细胞的增殖活性并促进细胞凋亡，为结直肠癌相关靶点治疗提供了体外实验依据。 |
| 英文摘要: |
| ABSTRACT Objective: To study the effect of targeting KRAS or TP53 mutation in SW620 cell line on cell proliferation activity by CRISPR/Cas9. Methods: First, we designed sgRNAs for mutated KRAS and TP53 in SW620 cells, and applied TIDE assay to detect their cleavage efficiency. Then, we performed cell proliferation assay to examine the change of proliferative activity after targeting KRAS or TP53 mutation in SW620 cells. Also, Annexin V-FITC/PI double staining was performed to detect apoptosis. Results: We constructed sgRNA plasmids targeting mutated KRAS and TP53 in SW620 cell line, respectively, and verified the endogenous cleavage efficiency of sgRNAs by TIDE assay. The proliferative capacity of SW620 cells was significantly diminished and the level of apoptosis was increased after targeting the mutated KRAS or TP53. Conclusion: In this study, mutated KRAS or TP53 gene in SW620 cells were edited by CRISPR/Cas9 technique. And we found that targeting either of these two mutations significantly inhibits the proliferative activity and promotes apoptosis in SW620 cells. This study provides further experimental evidence for targeted therapy of colorectal cancer. |
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