应用CRISPR-Cas9技术敲除ABCG2对结直肠癌细胞S1-M1-80的恶性生物学行为的研究
发布时间：2019-03-25 19:27
【摘要】：背景:CRISPR(Clustered Regularly Interspersed Short Palindromic Repeats)即规律间隔成簇短回文重复序列,它能抵御入侵的病毒和外源DNA,通过sg RNA引导Cas9核酸内切酶切割其定位的双链DNA,达到敲除的效果;ABCG2基因高表达与肿瘤多药耐药性密切相关,在癌细胞中过表达ABCG2将引起多耐药性导致治疗失败,然而却少有报道它在结直肠癌中除了多耐药性外和其他恶性生物行为的关系,本研究通过应用CRISPR-Cas9敲除ABCG2基因观察其对结直肠癌的多药耐药性和其他恶性生物行为的影响。方法:1、构建Lenti CRISPR v2载体;2、构建ABCG2敲除细胞株;3、MTT法检测稳定敲除细胞株对药物敏感性的改变;4、药物积累实验评估稳定敲除细胞株外排功能性的改变;5、通过MTT法、流式细胞术、划痕实验、Transwell侵袭实验、软琼脂糖克隆形成实验检测细胞生物行为的改变。结果:1、Lenti CRISPR v2载体构建成功;2、ABCG2稳定敲除细胞株构建成功;3、ABCG2基因敲除细胞株的药物敏感性提高;4、敲除ABCG2使结直肠癌细胞胞内药物积累增加;5、敲除ABCG2对结直肠癌细胞生长的影响不大,显著使细胞周期分布,迁移、侵袭克隆形成能力都减弱。结论:应用CRISPR-Cas9技术敲除ABCG2能抑制结直肠癌的恶性生物学行为。
[Abstract]:Background: CRISPR (Clustered Regularly Interspersed Short Palindromic Repeats) is a regularly spaced cluster of short palindromic repeats, which can resist invading viruses and exogenous DNA, to induce Cas9 endonucleases to cleave its localized double-stranded DNA, through sg RNA to achieve knockout effect. Overexpression of ABCG2 gene is closely related to multidrug resistance in cancer cells. Overexpression of ABCG2 in cancer cells may lead to treatment failure. However, there are few reports about its relationship with other malignant biological behaviors in colorectal cancer besides multidrug resistance. In this study, we observed the effect of CRISPR-Cas9 knockout ABCG2 gene on multidrug resistance and other malignant biological behaviors of colorectal cancer. Methods: 1, construction of Lenti CRISPR v2 vector; 2, construction of ABCG2 knockout cell line; 3, detection of drug sensitivity of stable knock-out cell line; 4, drug accumulation test to evaluate the functional changes of the exocytosis of stable knock-out cell line; 5. MTT assay, flow cytometry, scratch test, Transwell invasion test and soft agarose clone formation test were used to detect the changes of cell biological behavior. Results: 1, Lenti CRISPR v2 vector was successfully constructed; 2, ABCG2 stable knock-out cell line was successfully constructed; 3, ABCG2 gene knockout cell line was more sensitive to drugs; (4) knockout ABCG2 increased intracellular drug accumulation in colorectal cancer cells; 5. Knockout of ABCG2 had little effect on the growth of colorectal cancer cells, and significantly weakened the cell cycle distribution, migration and invasive clone formation ability. Conclusion: CRISPR-Cas9 knockout of ABCG2 can inhibit the malignant biological behavior of colorectal cancer.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.34
本文编号：2447247
[Abstract]:Background: CRISPR (Clustered Regularly Interspersed Short Palindromic Repeats) is a regularly spaced cluster of short palindromic repeats, which can resist invading viruses and exogenous DNA, to induce Cas9 endonucleases to cleave its localized double-stranded DNA, through sg RNA to achieve knockout effect. Overexpression of ABCG2 gene is closely related to multidrug resistance in cancer cells. Overexpression of ABCG2 in cancer cells may lead to treatment failure. However, there are few reports about its relationship with other malignant biological behaviors in colorectal cancer besides multidrug resistance. In this study, we observed the effect of CRISPR-Cas9 knockout ABCG2 gene on multidrug resistance and other malignant biological behaviors of colorectal cancer. Methods: 1, construction of Lenti CRISPR v2 vector; 2, construction of ABCG2 knockout cell line; 3, detection of drug sensitivity of stable knock-out cell line; 4, drug accumulation test to evaluate the functional changes of the exocytosis of stable knock-out cell line; 5. MTT assay, flow cytometry, scratch test, Transwell invasion test and soft agarose clone formation test were used to detect the changes of cell biological behavior. Results: 1, Lenti CRISPR v2 vector was successfully constructed; 2, ABCG2 stable knock-out cell line was successfully constructed; 3, ABCG2 gene knockout cell line was more sensitive to drugs; (4) knockout ABCG2 increased intracellular drug accumulation in colorectal cancer cells; 5. Knockout of ABCG2 had little effect on the growth of colorectal cancer cells, and significantly weakened the cell cycle distribution, migration and invasive clone formation ability. Conclusion: CRISPR-Cas9 knockout of ABCG2 can inhibit the malignant biological behavior of colorectal cancer.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.34
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