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

doi:10.3969/j.issn.1674-8115.2021.12.003

上海交通大学学报(医学版) ›› 2021, Vol. 41 ›› Issue (12): 1557-1563.doi: 10.3969/j.issn.1674-8115.2021.12.003

• 创新团队成果专栏 • 上一篇

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¹(), 俞思慧², 陆静³, 顾湘⁴, 张帆⁵, 程金科¹(), 王田实¹()

^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

出版日期:2021-09-23 发布日期:2021-09-23
通讯作者: 程金科,王田实 E-mail:yanyunhao@sjtu.edu.cn;jkcheng@shsmu.edu.cn;tianshi777@shsmu.edu.cn
作者简介:郝艳云（1991—），女，硕士生；电子信箱：yanyunhao@sjtu.edu.cn。
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)

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

Yan-yun HAO¹(), Si-hui YÜ², Jing LU³, Xiang GU⁴, Fan ZHANG⁵, Jin-ke CHENG¹(), Tian-shi WANG¹()

^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

Online:2021-09-23 Published:2021-09-23
Contact: Jin-ke CHENG,Tian-shi WANG E-mail:yanyunhao@sjtu.edu.cn;jkcheng@shsmu.edu.cn;tianshi777@shsmu.edu.cn
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)

摘要/Abstract

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

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

中图分类号:

R737.9

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.

Yan-yun HAO, Si-hui YÜ, Jing LU, Xiang GU, Fan ZHANG, Jin-ke CHENG, Tian-shi WANG. Role of SIRT3 SUMOylation deficiency in the proliferation and chemotherapeutic sensitivity of breast cancer cells MCF7[J]. JOURNAL OF SHANGHAI JIAOTONG UNIVERSITY (MEDICAL SCIENCE), 2021, 41(12): 1557-1563.

图/表 6

表1 sgRNA引物信息

Tab 1 Primers used for sgRNA

Primer	Sequence
SIRT3KO fwd1	5′-CACCGGGTCTTTGGCAGACTGTGC-3′
SIRT3KO rev1	5′-AAACGCACAGTCTGCCAAAGACCC-3′
SIRT3KO fwd2	5′-CACCGTACGATCTCCCGTACCCCG-3′
SIRT3KO rev2	5′-AAACCGGGGTACGGGAGATCGTAC-3′
SIRT3KO fwd3	5′-CACCGCCGCACGGCCTCGGTCAAGC-3′
SIRT3KO rev3	5′-AAACGCTTGACCGAGGCCGTGCGGC-3′

表2 构建反转录病毒质粒引物序列

Tab 2 Primers used for retroviral plasmids construction

Primer	Sequence
SIRT3WT fwd	5′-CGCGGATCCATGGCGTTCTGGGGTTGGCGC-3′
SIRT3WT rev	5′-CCGGAATTCTTTGTCTGGTCCATCAAGCTT-3′
SIRT3K288R fwd	5′-GGCGTTGTGAGGCCCGACATT-3′
SIRT3K288R rev	5′-AATGTCGGGCCTCACAACGCC-3′

图1 通过免疫沉淀和Western blotting鉴定构建的SIRT3 SUMO位点突变细胞系Note：A. MCF7-SIRT3KO lines identified by Western blotting (IB). B. Vector, SIRT3 WT and SIRT3 K288R cell lines identified by immunoprecipitation (IP) and Western blotting. SU-SIRT3—SUMOylated SIRT3.

Fig 1 Confirmation of SIRT3 SUMOylation deficiency cell line by immunoprecipitation and Western blotting

图2 3组细胞的7 d生长曲线和非黏附性乳腺球群细胞Note：A. Seven-day Growth curves of Vector, SIRT3 WT and SIRT3 K288R cells. B. Full-field views of single wells in the 96-well plates under microscopy (×100), and the values representing the diameters of the cell clumps.

Fig 2 Seven-day growth curves of cells and non-adherent breast cancer cell clumps in 3 groups

图3 3种细胞株经不同浓度阿霉素处理24 h（A）、48 h（B）和72 h（C）后的活性比较Note：①P=0.001, ②P=0.004, ③P=0.000, ④P=0.040, SIRT3 K288R vs SIRT3 WT.

Fig 3 Cell viability of 3 cell lines treated with various concentrations of ADM for 24 h (A), 48 h (B), and 72 h (C)

表3 阿霉素处理3种细胞不同时间的IC50（μg·mL-1）

Tab 3 IC50 of ADM in 3 cell lines for different time（μg·mL-1）

Time	Vector	SIRT3 WT	SIRT3 K288R
24 h	2.179	3.634	6.930
48 h	2.386	3.708	6.892
72 h	2.307	4.646	5.053

参考文献 32

1	Pavlova NN, Thompson CB. The emerging hallmarks of cancer metabolism[J]. Cell Metab, 2016, 23(1): 27-47.
2	Martinez-Outschoorn UE, Peiris-Pagés M, Pestell RG, et al. Cancer metabolism: a therapeutic perspective[J]. Nat Rev Clin Oncol, 2017, 14(1): 11-31.
3	Leone RD, Powell JD. Metabolism of immune cells in cancer[J]. Nat Rev Cancer, 2020, 20(9): 516-531.
4	Vyas S, Zaganjor E, Haigis MC. Mitochondria and cancer[J]. Cell, 2016, 166(3): 555-566.
5	Wang YP, Sharda A, Xu SN, et al. Malic enzyme 2 connects the Krebs cycle intermediate fumarate to mitochondrial biogenesis[J]. Cell Metab, 2021, 33(5): 1027-1041.e8.
6	Krug K, Jaehnig EJ, Satpathy S, et al. Proteogenomic landscape of breast cancer tumorigenesis and targeted therapy[J]. Cell, 2020, 183(5): 1436-1456.e31.
7	Carrico C, Meyer JG, He W, et al. The mitochondrial acylome emerges: proteomics, regulation by sirtuins, and metabolic and disease implications[J]. Cell Metab, 2018, 27(3): 497-512.
8	Hebert AS, Dittenhafer-Reed KE, Yu W, et al. Calorie restriction and SIRT3 trigger global reprogramming of the mitochondrial protein acetylome[J]. Mol Cell, 2013, 49(1): 186-199.
9	Inuzuka H, Gao D, Finley LW, et al. Acetylation-dependent regulation of Skp2 function[J]. Cell, 2012, 150(1): 179-193.
10	Dong XC, Jing LM, Wang WX, et al. Down-regulation of SIRT3 promotes ovarian carcinoma metastasis[J]. Biochem Biophys Res Commun, 2016, 475(3): 245-250.
11	Torrens-Mas M, Pons DG, Sastre-Serra J, et al. SIRT3 silencing sensitizes breast cancer cells to cytotoxic treatments through an increment in ROS production[J]. J Cell Biochem, 2017, 118(2): 397-406.
12	Bergaggio E, Riganti C, Garaffo G, et al. IDH2 inhibition enhances proteasome inhibitor responsiveness in hematological malignancies[J]. Blood, 2019, 133(2): 156-167.
13	Kim HS, Patel K, Muldoon-Jacobs K, et al. SIRT3 is a mitochondria-localized tumor suppressor required for maintenance of mitochondrial integrity and metabolism during stress[J]. Cancer Cell, 2010, 17(1): 41-52.
14	Li M, Chiang YL, Lyssiotis CA, et al. Non-oncogene addiction to SIRT3 plays a critical role in lymphomagenesis[J]. Cancer Cell, 2019, 35(6): 916-931.e9.
15	Cheng J, Bawa T, Lee P, et al. Role of desumoylation in the development of prostate cancer[J]. Neoplasia, 2006, 8(8): 667-676.
16	He J, Cheng J, Wang T. SUMOylation-mediated response to mitochondrial stress[J]. Int J Mol Sci, 2020, 21(16): 5657.
17	Seeler JS, Dejean A. SUMO and the robustness of cancer[J]. Nat Rev Cancer, 2017, 17(3): 184-197.
18	Wang T, Cao Y, Zheng Q, et al. SENP1-Sirt3 signaling controls mitochondrial protein acetylation and metabolism[J]. Mol Cell, 2019, 75(4): 823-834.e5.
19	He J, Shangguan X, Zhou W, et al. Glucose limitation activates AMPK coupled SENP1-Sirt3 signalling in mitochondria for T cell memory development[J]. Nat Commun, 2021, 12(1): 4371.
20	DeSantis CE, Ma J, Gaudet MM, et al. Breast cancer statistics, 2019[J]. CA Cancer J Clin, 2019, 69(6): 438-451.
21	Harbeck N, Penault-Llorca F, Cortes J, et al. Breast cancer[J]. Nat Rev Dis Primers, 2019, 5(1): 66.
22	Jabbour E, Ravandi F, Kebriaei P, et al. Salvage chemoimmunotherapy with inotuzumab ozogamicin combined with mini-hyper-CVD for patients with relapsed or refractory Philadelphia chromosome-negative acute lymphoblastic leukemia: a phase 2 clinical trial[J]. JAMA Oncol, 2018, 4(2): 230-234.
23	Horwitz S, O'Connor OA, Pro B, et al. Brentuximab vedotin with chemotherapy for CD30-positive peripheral T-cell lymphoma (ECHELON-2): a global, double-blind, randomised, phase 3 trial[J]. Lancet, 2019, 393(10168): 229-240.
24	Cesarman E, Damania B, Krown SE, et al. Kaposi sarcoma[J]. Nat Rev Dis Primers, 2019, 5(1): 9.
25	Basho RK, Gilcrease M, Murthy RK, et al. Targeting the PI3K/AKT/mTOR pathway for the treatment of mesenchymal triple-negative breast cancer: evidence from a phase 1 trial of mTOR inhibition in combination with liposomal doxorubicin and bevacizumab[J]. JAMA Oncol, 2017, 3(4): 509-515.
26	Cai L, Tu J, Song L, et al. Proteome-wide mapping of endogenous SUMOylation sites in mouse testis[J]. Mol Cell Proteomics, 2017, 16(5): 717-727.
27	Vander Heiden MG, Cantley LC, Thompson CB. Understanding the Warburg effect: the metabolic requirements of cell proliferation[J]. Science, 2009, 324(5930): 1029-1033.
28	Guri Y, Colombi M, Dazert E, et al. mTORC2 promotes tumorigenesis via lipid synthesis[J]. Cancer Cell, 2017, 32(6): 807-823.e12.
29	Bogachek MV, Chen Y, Kulak MV, et al. Sumoylation pathway is required to maintain the basal breast cancer subtype[J]. Cancer Cell, 2014, 25(6): 748-761.
30	Kessler JD, Kahle KT, Sun T, et al. A SUMOylation-dependent transcriptional subprogram is required for Myc-driven tumorigenesis[J]. Science, 2012, 335(6066): 348-353.
31	Morris JR, Boutell C, Keppler M, et al. The SUMO modification pathway is involved in the BRCA1 response to genotoxic stress[J]. Nature, 2009, 462(7275): 886-890.
32	Madeddu C, Gramignano G, Floris C, et al. Role of inflammation and oxidative stress in post-menopausal oestrogen-dependent breast cancer[J]. J Cell Mol Med, 2014, 18(12): 2519-2529.

[1]	周天浩, 辛肇晨, 杜少倩, 曹源, 许静轩, 劳曾红, 王红霞. 乳腺癌类器官共培养技术的建立和优化[J]. 上海交通大学学报(医学版), 2021, 41(8): 1017-1024.
[2]	季艳杰, 罗浩, 蔡海燕, 刘欣宇, 金诗佳, 粟深月, 徐含章, 雷虎, 吴英理. CDDO-Me对三阴性乳腺癌细胞泛素特异性蛋白酶2a活性及细胞增殖的抑制作用[J]. 上海交通大学学报(医学版), 2021, 41(8): 1025-1032.
[3]	王成志, 邓华云, 庞智, 黄雷. MUC1在HER2阳性乳腺癌发病中的作用及机制研究[J]. 上海交通大学学报(医学版), 2021, 41(7): 839-848.
[4]	徐建华, 江萍, 邓炯. ATP结合盒蛋白G超家族成员2在肺癌中的表达及意义[J]. 上海交通大学学报(医学版), 2021, 41(6): 830-833.
[5]	张海燕, 储晓英. 非阿片类镇痛药应用在全身麻醉喉罩置入的乳腺癌保乳手术中的可行性[J]. 上海交通大学学报(医学版), 2021, 41(5): 637-641.
[6]	张佳玲, 张凤春, 徐迎春. 乳腺癌脑转移系统治疗的研究进展[J]. 上海交通大学学报(医学版), 2021, 41(5): 671-677.
[7]	赵晓军, 乔志刚, 梁挺宇, 安艳玲. 乳腺癌易感基因1蛋白在脑胶质瘤中的表达及对替莫唑胺敏感性的影响[J]. 上海交通大学学报(医学版), 2021, 41(1): 118-122.
[8]	陈思，刘春良，赵倩，孙海鹏，刘云霞. 基于生存分析的生物信息学方法筛选乳腺癌枢纽基因和关键通路[J]. 上海交通大学学报（医学版）, 2020, 40(3): 294-.
[9]	叶欣，周晓云，杨莉，王杰，何奇. 不同年龄范围界定下的年轻乳腺癌患者的临床病理特征及预后分析[J]. 上海交通大学学报（医学版）, 2020, 40(3): 351-.
[10]	王钰婷1, 2，刘锦燕2，史册1，赵珺涛1, 2，项明洁1, 2. 白念珠菌ERG3基因敲除及其对耐药性的影响[J]. 上海交通大学学报（医学版）, 2020, 40(2): 163-.
[11]	程龙1，徐五琴2，张鹏1，黄建军1，李小宁1. 下调miR-27a表达对三阴乳腺癌细胞增殖、迁移和侵袭的影响[J]. 上海交通大学学报（医学版）, 2020, 40(2): 188-.
[12]	张凤春1, 2，张硕渊3，陈天恩3，徐迎春3. PD-1/PD-L1通路在三阴性乳腺癌预后预测及治疗中的意义[J]. 上海交通大学学报（医学版）, 2020, 40(1): 128-.
[13]	何沁，张伟滨，沈宇辉. 乳腺癌脊柱转移患者预后预测模型的构建[J]. 上海交通大学学报(医学版), 2020, 40(09): 1243-1248.
[14]	陆裕杰，金泽宇，李亚芬，沈坤炜，陈伟国，陈小松. 乳腺癌局部区域复发病灶受体状态检测及对后续治疗的指导价值[J]. 上海交通大学学报（医学版）, 2019, 39(9): 1071-.
[15]	来星，方超. 基于光诱导的粒径可变阿霉素纳米递送系统[J]. 上海交通大学学报（医学版）, 2019, 39(4): 347-.

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

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

RichHTML

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

图/表 6

参考文献 32

相关文章 15

编辑推荐

Metrics