基于CRISPR/Cas9-SAM系统<i>CHD5</i>基因过表达慢病毒载体的构建及对膀胱癌T24细胞增殖,迁移和侵袭能力的影响 <sup>*</sup>

doi:10.13523/j.cb.1908028

中国生物工程杂志

2020, Vol. 40

Issue (3): 1-8 DOI: 10.13523/j.cb.1908028

研究报告

基于CRISPR/Cas9-SAM系统CHD5基因过表达慢病毒载体的构建及对膀胱癌T24细胞增殖,迁移和侵袭能力的影响 ^*

黄胜^1,^**,严启滔^2,^**,熊仕琳³,彭弈骐¹,赵蕊^3,^***(

)

1 南方医科大学珠江医院广州 510282
2 南部战区总医院老年感染与器官功能支持重点实验室广州 510010
3 南方医科大学基础医学院生物化学与分子生物学教研室广州 510515

Construction of CHD5 Gene Overexpressing Lentiviral Vector Based on CRISPR/Cas9-SAM System and the Effect of CHD5 on Proliferation, Migration and Invasion in T24 Cells

HUANG Sheng^1,^**,YAN Qi-tao^2,^**,XIONG Shi-lin³,PENG Yi-qi¹,ZHAO Rui^3,^***(

)

1 Zhujiang Hospital of Southern Medical University,Guangzhou 510282,China
2 General Hospital of Southern Theatre Command Key Laboratory of Geriatric Infection and Organ Function Support,Guangzhou 510010,China
3 Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Southern Medical University 510515,China

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摘要：

目的利用CRISPR/Cas9-SAM系统构建CHD5基因过表达慢病毒载体,并分析其对膀胱癌细胞T24增殖,迁移和侵袭能力的影响.方法: 针对CHD5基因设计3个sgRNA(sgRNA-1,sgRNA-2,sgRNA-3),将sgRNA连入LV-sgRNA-MS2-P65-HSF1-Neo载体,经293T细胞包装后获得高滴度慢病毒颗粒.病毒以MOI=10感染膀胱癌细胞T24.RT-qPCR和Western blot分别检测感染病毒后T24细胞CHD5 mRNA和蛋白表达水平,CCK8实验,流式分析,划痕实验和Transwell实验检测CHD5过表达对T24细胞增殖,凋亡,迁移和侵袭能力的影响.结果: 成功构建CHD5过表达慢病毒载体.慢病毒感染T24细胞后,RT-qPCR和Western blot证实,T24细胞CHD5的mRNA和蛋白表达水平显著高于空白组和阴性对照组(P<0.05),并且sgRNA-3-MS2-P65-HSF1序列的作用最为显著.CCK8及流式分析结果显示,过表达CHD5抑制T24细胞增殖,促进凋亡,与对照组相比,均有统计学意义(P<0.001).划痕和Transwell实验结果表明,过表达CHD5可抑制T24细胞迁移和侵袭能力(P<0.01).结论: 成功构建CHD5过表达慢病毒.过表达CHD5能促进膀胱癌细胞T24凋亡,抑制其增殖,迁移和侵袭能力.

关键词： CHD5; CRISPR/Cas9-SAM系统; 膀胱癌; 慢病毒

Abstract:

Objective: To construct a CHD5 gene overexpressing lentiviral vector using CRISPR/Cas9-SAM system and analyze its effect on the proliferation, migration and invasion in bladder cancer cell line T24.Methods: Three pairs of sgRNAs (sgRNA1, sgRNA-2, sgRNA-3) were designed targeting CHD5 gene. The sgRNA was inserted into the LV-sgRNA-MS2-P65-HSF1-Neo vector, and the lentivirus particles with high titer were obtained after 293T cell packaging. Subsequently, bladder cancer cell line T24 were infected with lentivirus at MOI=10. The expression of CHD5 mRNA and protein in T24 cells was examined by RT-qPCR and Western blot, respectively. CCK8 assay, flow cytometry, wound healing and transwell assay were performed to detect the effects of overexpression of CHD5 on the proliferation, apoptosis, migration and invasion of T24 cells.Results: The CHD5 overexpressing lentiviral vector was successfully constructed. The results of RT-qPCR and Western blot confirmed that the mRNA and protein expression of CHD5 in T24 cells were significantly higher than those in the blank and negative control groups after lentivirus infection(P<0.05), and the effect of sgRNA-3-MS2-P65-HSF1 was the most significant.CCK8 and flow cytometry analysis showed that overexpression of CHD5 inhibited the proliferation and promoted apoptosis of T24 cells, which had statistical significance compared with the control group (P<0.001). Wound healing and transwell assay showed that overexpression of CHD5 inhibited the migration and invasion of T24 cells (P<0.01).Conclusion: CHD5-SAM lentivirus has been successfully constructed. Overexpression of CHD5 could induce apoptosis, inhibit proliferation, migration and invasion of bladder cancer cell line T24.

Key words: CHD5 CRISPR/Cas9-SAM system Bladder cancer Lentivirus

收稿日期: 2019-08-15 出版日期: 2020-04-18

ZTFLH:

Q819

基金资助: * 广州市科技计划项目珠江科技新星专项(201610010174);国家自然科学基金面上项目(81771710);广州市科技计划项目(20170701006)

通讯作者: 赵蕊 E-mail: zhaoruiruizhao@qq.com

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引用本文:

黄胜, 严启滔, 熊仕琳, 彭弈骐, 赵蕊. 基于CRISPR/Cas9-SAM系统CHD5基因过表达慢病毒载体的构建及对膀胱癌T24细胞增殖,迁移和侵袭能力的影响 ^*[J]. 中国生物工程杂志, 2020, 40(3): 1-8.

HUANG Sheng, YAN Qi-tao, XIONG Shi-lin, PENG Yi-qi, ZHAO Rui. Construction of CHD5 Gene Overexpressing Lentiviral Vector Based on CRISPR/Cas9-SAM System and the Effect of CHD5 on Proliferation, Migration and Invasion in T24 Cells. China Biotechnology, 2020, 40(3): 1-8.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.1908028 或 https://manu60.magtech.com.cn/biotech/CN/Y2020/V40/I3/1

表2 CHD5和β-Actin RT-qPCR实验引物序列

图1 CHD5-sgRNA表达载体测序结果

图2 SAM双载体病毒感染T24细胞后CHD5的表达水平

图3 CCK检测细胞增殖能力

图4 过表达CHD5对细胞周期和凋亡的影响

图5 CHD5对T24细胞迁移和侵袭能力的影响(×100)

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