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


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