| 刘清桂,苏 静,陈佳佳.下调SIRT6对肝癌干细胞干性维持的抑制作用[J].中国肿瘤,2020,29(7):544-553. |
| 下调SIRT6对肝癌干细胞干性维持的抑制作用 |
| Effect of SIRT6 Silencing on Stemness Maintaining of Hepatic Cancer Stem Cells |
| 中文关键词 修订日期：2020-04-17 |
| DOI：10.11735/j.issn.1004-0242.2020.07.A012 |
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| 中文关键词: 组蛋白去乙酰化酶 SIRT6 肝癌干细胞 Nanog 干细胞特性 衰老 |
| 英文关键词:histone deacetylase SIRT6 hepatic cancer stem cells Nanog stemness senescence |
| 基金项目:国家自然科学基金青年科学基金项目（31601101）；全军医学科技青年培育计划项目（17QNP11） |
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| 中文摘要: |
| 摘 要：［目的］ 探讨抑制SIRT6表达在肝癌干细胞干性维持中的作用及潜在机制。［方法］ 在不同肝癌细胞系中构建肝癌干细胞模型（Lv-Pnanog-GFP），利用流式细胞术分选出不同来源的肝癌干细胞，采用蛋白免疫印迹（Western blot，WB）和免疫细胞化学（immunocytochemistry，ICC）技术检测SIRT6蛋白在肝癌干细胞中的表达水平；构建SIRT6慢病毒短发卡RNA感染载体（shSIRT6）感染肝癌干细胞，采用WB技术检测shSIRT6的干扰效率；采用实时定量反转录聚合酶链反应（real-time PCR）检测抑制SIRT6后肝癌干细胞的干性基因（Nanog、OCT4、CD13、SOX2、EpCAM、CD44）mRNA的表达水平，ICC方法检测EpCAM蛋白的表达水平；采用5-乙炔基-2’脱氧尿嘧啶核苷（EdU）试剂盒和细胞增殖活力（CCK8）试剂盒检测抑制SIRT6表达后肝癌干细胞的增殖能力；采用克隆形成和成球实验检测抑制SIRT6后肝癌干细胞的自我更新能力；利用衰老相关β-半乳糖苷酶（SA-β-Gal）试剂盒检测抑制SIRT6后肝癌干细胞的衰老水平。［结果］ WB和ICC检测结果显示，Nanog阳性肝癌干细胞中SIRT6表达水平明显高于Nanog阴性肝癌细胞；real-time PCR检测结果显示，抑制SIRT6后，肝癌干细胞的干性基因（Nanog、OCT4、CD13、SOX2、EpCAM、CD44）mRNA表达水平降低。与real-time PCR结果一致，ICC结果也表明，抑制SIRT6后，干性标志物EpCAM的蛋白表达水平降低（P<0.01）。细胞增殖检测结果进一步显示，抑制SIRT6，肝癌干细胞的增殖水平降低（P<0.05）。克隆形成实验和细胞成球实验结果显示，抑制SIRT6导致肝癌干细胞的克隆形成能力和成球能力一致性降低（P<0.01）。SA-β-Gal活性检测结果显示，抑制SIRT6后，肝癌干细胞的SA-β-Gal活性显著性提高（P<0.01）。［结论］肝癌干细胞高表达SIRT6，通过抑制SIRT6表达可诱导肝癌干细胞发生衰老，导致其干性标志物表达水平下降，增殖能力降低，肝癌干细胞克隆形成和成球能力下降。 |
| 英文摘要: |
| Abstract：［Purpose］ To investigate the role of sirtuins6 (SIRT6) on the stemness maintaining of hepatic cancer stem cells. ［Methods］Liver cancer stem cells (LCSCs) in human hepatocellular carcinoma cell lines were identified with Lv-Pnanog-GFP model. The expression of SIRT6 in purified LCSCs was detected by Western blot and immunocytochemistry method. Lentivirus of SIRT6-specific siRNA was packaged and transfected into LCSCs，the expression of SIRT6 in LCSCs was detected by Western blotting. Real-time PCR was used to detect mRNA expression of hepatic cancer stem cells stemness markers (Nanog，OCT4，CD13，SOX2，EpCAM and CD44) in LCSCs，immunocytochemistry method was used to detect the protein expression of EpCAM. The proliferation ability of LCSCs was investigated by EdU cell proliferation kit and cell counting kit-8，and the self-renewal ability of LCSCs was investigated by colony formation assay and sphere formation assay. The SA-β-Gal assay was used to evaluated the senescence of LCSCs. ［Results］ The expression of SIRT6 in LCSCs was significantly higher than that in non-LCSCs. Real-time PCR revealed that the expression of genes involved in stemness（Nanog，OCT4，CD13，SOX2，EpCAM，CD44） was significantly decreased when SIRT6 was knockdown by shRNA. Immunocytochemistry displayed that the EpCAM protein was downreguated compared with control. In addition，EdU incorporation assay and CCK8 assay revealed that the proliferating LCSCs were significantly decreased in SIRT6 silencing (P<0.05). Consistently，SIRT6 knockdown inhibited the self-renewal ability of LCSCs，the rate of colony formation and the percentage of sphere formation was markedly decreased compared to shScrambled control (P<0.01). Moreover，the SA-β-Gal activity was markedly enhanced (P<0.01).［Conclusion］ SIRT6 is upregulated in LCSCs. SIRT6 knockdown can suppress the expression of stemness-related makers，inhibit the growth of LCSCs and downregulate self-renewal ability by inducing cellular senescence. |
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