桑辛素对喉癌干细胞干性表型调控的作用研究

陈辰; 许子寒; 王力

doi:10.12182/20200960503

桑辛素对喉癌干细胞干性表型调控的作用研究

The Effect of Morusin on Stemness Phenotype of Laryngeal Cancer Stem Cell

摘要

摘要:
目的探索桑辛素对喉癌干细胞干性表型调控的影响。
方法流式细胞仪分选并检测CD133⁺喉癌干细胞比例；肿瘤球形成实验检测CD133⁺喉癌干细胞的自我更新能力；Transwell实验检测CD133⁺喉癌干细胞的迁移能力；改良MTT实验检测化疗药物对CD133⁺喉癌干细胞的细胞毒性作用；免疫荧光染色、实时荧光定量PCR（RT-qPCR）及Western blot检测CD133⁺喉癌干细胞的干细胞标志物表达；在不同浓度的桑辛素处理CD133⁺喉癌干细胞后（以桑辛素0 μmol/L为对照），通过肿瘤球形成实验、Transwell实验、改良MTT实验及Western blot，分别检测CD133⁺喉癌干细胞的自我更新能力、迁移能力、化疗药物的细胞毒性作用及干细胞标志物表达变化。
结果流式细胞仪分选结果显示，CD133⁺喉癌干细胞占喉癌细胞的比例为（3.50±0.34）%；经过培养富集后，其比例可达（93.20±5.23）%。肿瘤球形成实验结果显示，与喉癌细胞相比，CD133⁺喉癌干细胞具有增强的自我更新能力 (P<0.001)；Transwell实验显示，与喉癌细胞相比，CD133⁺喉癌干细胞的迁移能力增强（P<0.05）；改良MTT实验结果显示，与喉癌细胞相比，CD133⁺喉癌干细胞抵抗化疗药物（5-氟尿嘧啶及顺铂）的细胞毒性作用（P<0.05）；免疫荧光染色、RT-qPCR及Western blot结果显示，干细胞标志物（CD133、ALDH1、Sox2、ABCG2及N-cadherin）在CD133⁺喉癌干细胞中呈高表达水平。通过不同浓度的桑辛素处理CD133⁺喉癌干细胞，其自我更新能力降低（P<0.05）；迁移能力亦下降（P<0.05）；此外，桑辛素处理的CD133⁺喉癌干细胞对化疗药物的细胞毒性作用更加敏感（P<0.05）；Western blot结果显示，不同浓度的桑辛素处理的CD133⁺喉癌干细胞，其上述的干细胞标志物表达水平下调（P<0.05）。
结论 CD133⁺喉癌干细胞具有干性表型特征；桑辛素可减弱喉癌干细胞的干性表型，可能与下调其干细胞标志物表达相关。

Abstract:
Objective To investigate the regulation effect of Morusin on stemness phenotype of laryngeal cancer stem cells.
Methods Separation and detection the proportion of CD133⁺ laryngeal cancer stem cells through flow cytometry; evaluation the self-renewal ability of CD133⁺ laryngeal cancer stem cells by tumor sphere formation assay; exploring the migration ability of CD133⁺ laryngeal cancer stem cells by Transwell assay; analyzing the cytotoxicity of chemotherapy drugs on CD133⁺ laryngeal cancer stem cells by modified MTT assay; detection of the expression levels of stemness associated markers by immunofluorescence staining, RT-qPCR and Western blot. After treatment with different concentrations of Morusin, cells were performed the above experiments for detection the self-renewal ability, migration ability, cytotoxicity resistance and expression of stemness associated markers.
Results Flow cytometry analysis showed that the proportion of CD133⁺ laryngeal cancer stem cells was (3.50±0.34)%, while after enrichment, the proportion increased to (93.20±5.23)%. CD133⁺ laryngeal cancer stem cells exhibited better self-renewal ability (P<0.001) and migratory ability (P<0.05); they were resistant to the cytotoxicity of chemotherapy drug (P<0.05), and highly expressed of stemness associated markers. After being treated with Morusin, the self-renewal and migratory abilities of CD133⁺ laryngeal cancer stem cells were reduced (P<0.05). In addition, after treated with Morusin, CD133⁺laryngeal cancer stem cells were more sensitive to chemotherapy drugs; moreover, the expression levels of stemness associated markers were decreased.
Conclusion CD133⁺ laryngeal cancer stem cells possessed stemness phenotypic characteristics. Morusin attenuated stemness phenotype of laryngeal cancer stem cells, which may be related to its down-regulation effect on stemness associated markers.

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