上海交通大学学报（医学版）

上海交通大学学报（医学版） >

2021 , Vol. 41 >Issue 12: 1557 - 1563

DOI: https://doi.org/10.3969/j.issn.1674-8115.2021.12.003

创新团队成果专栏

SIRT3去SUMO化修饰调节乳腺癌细胞MCF7增殖及化疗药物敏感性的研究

  • 郝艳云 ,
  • 俞思慧 ,
  • 陆静 ,
  • 顾湘 ,
  • 张帆 ,
  • 程金科 ,
  • 王田实
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  • 1.上海交通大学基础医学院生物化学与分子细胞生物学系，上海 200025
    2.上海交通大学附属第一人民医院妇产科，上海 201620
    3.上海交通大学医学院附属精神卫生中心物质成瘾科，上海 200030
    4.上海交通大学医学院附属第九人民医院眼科，上海 200011
    5.上海交通大学医学院附属新华医院耳鼻咽喉头颈外科，上海 200092
郝艳云（1991—），女，硕士生；电子信箱：yanyunhao@sjtu.edu.cn。
程金科，电子信箱：jkcheng@shsmu.edu.cn。#为共同通信作者。
王田实，电子信箱：tianshi777@shsmu.edu.cn

网络出版日期: 2021-01-28

基金资助

国家自然科学基金(81730082);上海交通大学“科技创新专项资金”项目(19X160010017);上海交通大学医学院高水平地方高校创新团队(SSMU-ZLCX20180102)

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Role of SIRT3 SUMOylation deficiency in the proliferation and chemotherapeutic sensitivity of breast cancer cells MCF7

  • Yan-yun HAO ,
  • Si-hui Yü ,
  • Jing LU ,
  • Xiang GU ,
  • Fan ZHANG ,
  • Jin-ke CHENG ,
  • Tian-shi WANG
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  • 1.Department of Biochemistry and Molecular Cell Biology, Shanghai Jiao Tong University College of Basic Medical Sciences, Shanghai 200025, China
    2.Department of Obstetrics and Gynecology, Shanghai General Hospital, Shanghai Jiao Tong University, Shanghai 201620, China
    3.Department of Substance Addiction, Shanghai Mental Health Center, Shanghai Jiao Tong University School of Medicine, Shanghai 200030, China
    4.Department of Ophthalmology, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200011, China
    5.Department of Otolaryngology Head and Neck Surgery, Xinhua Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200092, China
CHENG Jin-ke, E-mail: jkcheng@shsmu.edu.cn. #Co-corresponding authors.
WANG Tian-shi, E-mail: tianshi777@shsmu.edu.cn

Online published: 2021-01-28

Supported by

National Natural Science Foundation of China(81730082);Special Funding Project of Shanghai Jiao Tong University(19X160010017);Innovative Research Team of High-Level Local Universities in Shanghai(SSMU-ZLCX20180102)

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摘要

目的·研究沉默调节蛋白3（sirtuin 3，SIRT3）去SUMO化修饰对乳腺癌细胞的增殖和耐药性的影响。方法·用CRISPR-Cas9质粒pX330-sgRNA敲除MCF7乳腺癌细胞系SIRT3基因，构建SIRT3KO的MCF7细胞系；在此细胞系基础上，利用表达SIRT3 K288R（SUMO化修饰位点突变型）和SIRT3 WT（野生型）的反转录病毒，以及空载对照（Vector）病毒进行感染，以构建SIRT3 SUMO化修饰位点突变的细胞系及其对照细胞系。3种细胞株在相同条件下培养后进行细胞计数并在显微镜下进行观察，确定细胞数量和形态的变化；用不同浓度的阿霉素分别处理细胞24、48、72 h，检测3种细胞株的耐药程度。结果·成功构建了敲除SIRT3的MCF7细胞系，并利用反转录病毒感染构建了表达SIRT3 K288R和SIRT3 WT以及空载对照的细胞系；通过细胞计数发现，在相同培养时间下，SIRT3 K288R细胞系的增殖速率显著低于SIRT3 WT和Vector细胞系（P=0.000）；在显微镜下测量非黏附性乳腺球群细胞的大小，发现SIRT3 K288R细胞系的直径较SIRT3 WT和Vector细胞系小，增殖缓慢。阿霉素耐药实验结果显示，在细胞培养24、48和72 h时，不同阿霉素浓度（1.25、2.5、5和10 μg/mL）处理下的SIRT3 K288R细胞系的活性均显著高于SIRT3 WT和Vector细胞系（均P<0.05）；并且相比另外2个细胞系，阿霉素在SIRT3 K288R细胞系中的半数抑制浓度（IC50）也较高。结论·去SUMO化修饰的SIRT3能抑制乳腺癌细胞MCF7的增殖，但增加其对阿霉素的耐药性。

关键词： 沉默调节蛋白3; SUMO化修饰; 乳腺癌; 阿霉素; 耐药性

本文引用格式

郝艳云 , 俞思慧 , 陆静 , 顾湘 , 张帆 , 程金科 , 王田实 . SIRT3去SUMO化修饰调节乳腺癌细胞MCF7增殖及化疗药物敏感性的研究[J]. 上海交通大学学报（医学版）, 2021 , 41(12) : 1557 -1563 . DOI: 10.3969/j.issn.1674-8115.2021.12.003

Abstract

Objective

·To investigate the effects of deSUMOylation of sirtuin 3 (SIRT3) on the proliferation and chemotherapeutic drug resistance in breast cancer cells.

Methods

·In order to construct the MFC7-SIRT3KO cell line, the CRISPR-Cas9 plasmid pX330-sgRNA targeting SIRT3 gene was designed. Then, this cell line was infected with SIRT3 K288R (sumoylation modification site mutant), SIRT3 WT (wild type) or Vector retrovirus to establish the SIRT3 SUMOylation site mutation and control cell lines. Cell counting and microscopic observation were used to determine the changes in tumor cell number and morphology under the same incubation condition. To detect the chemotherapeutic drug resistance of SIRT3 K288R and the control cells, these cells were treated with different concentrations of adriamycin (ADM) for 24 h, 48 h and 72 h.

Results

·The MFC7-SIRT3KO cell line was constructed successfully, and then SIRT3 K288R, SIRT3 WT and Vector cell lines were established. The cell growth curves showed that the growth rate of SIRT3 K288R cells was significantly slower than those of SIRT3 WT and Vector cells under the same incubation condition (P=0.000). This phenotype was also observed under the microscope. The diameter of non-adherent breast cancer cell clumps of SIRT3 K288R cells was smaller than those of the control cells. ADM resistance experiment showed that the activity of SIRT3 K288R cells was significantly higher than those of Vector and SIRT3 WT cells treated with different concentrations of ADM (1.25 μg/mL, 2.5 μg/mL, 5 μg/mL and 10 μg/mL) for 24 h, 48 h and 72 h (P<0.05), and the half maximal inhibitory concentration (IC50) of ADM in the SIRT3 K288R cells was higher than those in the control cells.

Conclusion

·The SIRT3 SUMOylation deficiency inhibits the proliferation of breast cancer cells MCF7, while increases the drug resistance to ADM.

Key words： sirtuin 3 (SIRT3); SUMOylation; breast cancer; adriamycin (ADM); drug resistance

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