沉默YAP基因提高伊马替尼耐药的人髓性白血病细胞K562药物敏感性的研究

doi:10.12092/j.issn.1009-2501.2018.09.007

中国临床药理学与治疗学 ›› 2018, Vol. 23 ›› Issue (9): 1008-1014.doi: 10.12092/j.issn.1009-2501.2018.09.007

沉默YAP基因提高伊马替尼耐药的人髓性白血病细胞K562药物敏感性的研究

王瑾¹，杨毅¹，官俏兵²，陈启绪³，郭丽²，韩晨阳¹

¹嘉兴市第二医院药学部，²中心实验室，嘉兴 314001，浙江；³山东省科学院，济南 250000，山东

收稿日期:2018-05-03 修回日期:2018-05-30 出版日期:2018-09-26 发布日期:2018-09-26
通讯作者: 韩晨阳，男，硕士，药师，研究方向：药理学。 E-mail：691513770@qq.com
作者简介:王瑾，女，本科，主管药师，研究方向：药理学。 E-mail：wt198805@126.com
基金资助:
浙江省科技厅实验动物项目（2017C37174）；浙江省卫生厅面上项目（2018KY804）

YAP gene silencing to improve the drug sensitivity of imatinib resistant myeloid leukemia cells K562

WANG Jin¹, YANG Yi¹, GUAN Qiaobin², CHEN Qixu³, GUO Li ², HAN Chenyang¹

¹Pharmacy Department, ²Central Laboratory, Second Hospital of Jiaxing, Jiaxing 314001, Zhejiang, China; ³Academy of Sciences of Shandong Province, Jinan 250000, Shandong, China

Received:2018-05-03 Revised:2018-05-30 Online:2018-09-26 Published:2018-09-26

摘要/Abstract

摘要：

目的: 探究沉默YAP基因后，提高伊马替尼耐药的K562细胞（K562/IMA）对于伊马替尼敏感性的作用及相关机制，为伊马替尼耐药的人髓性白血病治疗提供参考。方法: 采用小干扰RNA技术（siRNA）转染YAP的siRNA-YAP沉默K562/IMA的YAP基因。设置对照组（Control）、siRNA阴性对照组（siRNA-NC）和YAP siRNA组（siRNA-YAP）。采用RT-qPCR和Western blot法鉴定沉默后各组细胞YAP蛋白和mRNA的表达水平。CCK-8法检测各组细胞对于伊马替尼的敏感性，并计算半数抑制浓度IC50。流式细胞术检测各组细胞的凋亡率。Western blot法检测YAP、Bcl-2、Bax、cleaved-caspase-3、caspase-3、细胞色素C（Cyto-C）的表达水平，Transwell小室检测细胞转移侵袭能力。结果: YAP基因沉默后，siRNA-YAP组细胞中YAP的蛋白和mRNA表达水平显著低于Control组和siRNA-NC组，沉默效果较好。siRNA-YAP的IC50值显著低于Control组和siRNA-NC组，具有统计学意义（P<0.05），而流式细胞术结果显示，siRNA-YAP组细胞在相同药物剂量下，凋亡率显著高于Control组和siRNA-NC组，具有统计学意义（P<0.05）。YAP沉默的K562/IMA细胞迁移侵袭能力显著下降。伊马替尼干预后，siRNA-YAP细胞中Bcl-2/Bax的水平下调，cleaved-caspase-3、caspase-3和Cyto-C的水平上调，相比Control组和siRNA-NC组，具有统计学意义（P<0.05）。结论: 沉默伊马替尼耐药的K562/IMA细胞中YAP基因，可以恢复肿瘤细胞对于伊马替尼的敏感性，可以促进线粒体凋亡途径的激活，导致耐药肿瘤细胞的凋亡。

关键词: YAP基因, 伊马替尼, 人髓性白血病细胞, 敏感性

Abstract:

AIM: To explore the effect and mechanism of enhanced imatinib resistant K562 cells (K563/IMA) on imatinib sensitivity after YAP gene silencing, and to support the treatment of imatinib resistant myeloid leukemia. METHODS: Transfection of YAP gene silencing with siRNA-YAP was conducted by small interference RNA technique (siRNA) in K562/IMA. The control group (control), the siRNA negative control group (siRNA-NC) and the YAP siRNA group (siRNA-YAP) were established. The expression level of YAP protein and mRNA in each cell after silencing was identified by RT-QPCR and Western blot. The sensitivity of each cell to imatinib was detected by CCK-8, and the median inhibitory concentration of IC50 was calculated. Flow cytometry was used to detect the apoptosis rate of each group. The expression level of YAP, Bcl-2, Bax, cleaved-caspase3, caspase-3 and Cyto-C were detected by Western blot. The metastasis and invasion were detected by Transwell compartment. RESULTS: After the YAP gene silenced, the expression level of YAP protein and mRNA in the siRNA-YAP was significantly lower than that in the control and the siRNA-NC. The IC50 value of siRNA-YAP was significantly lower than that of control and siRNA-NC, which was statistically significant (P<0.05). The results of flow cytometry showed that the apoptotic rate of siRNA-YAP was significantly higher than that of control and siRNA-NC at the same dosage, which was statistically significance (P<0.05). The migration and invasiveness of K562/IMA cells in YAP silenced were decreased significantly. After imatinib intervene, the level of Bcl-2/Bax in siRNA-YAP cells was down-regulated, and the levels of cleaved-caspase3, caspase-3 and Cyto-C were up-regulated. Compared with control and siRNA-NC, it was statistically significant (P<0.05). CONCLUSION: To silence the YAP gene in imatinib resistant K562/IMA cells can restore the sensitivity of tumor cells to imatinib, and promote the activation of mitochondrial apoptotic pathway, resulting in the apoptosis of drug-resistant tumor cells.

Key words: YAP gene, imatinib, human myeloid leukemia cells, sensitivity

中图分类号:

王瑾，杨毅，官俏兵，陈启绪，郭丽，韩晨阳. 沉默YAP基因提高伊马替尼耐药的人髓性白血病细胞K562药物敏感性的研究[J]. 中国临床药理学与治疗学, 2018, 23(9): 1008-1014.

WANG Jin, YANG Yi, GUAN Qiaobin, CHEN Qixu, GUO Li, HAN Chenyang. YAP gene silencing to improve the drug sensitivity of imatinib resistant myeloid leukemia cells K562[J]. Chinese Journal of Clinical Pharmacology and Therapeutics, 2018, 23(9): 1008-1014.

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沉默YAP基因提高伊马替尼耐药的人髓性白血病细胞K562药物敏感性的研究

YAP gene silencing to improve the drug sensitivity of imatinib resistant myeloid leukemia cells K562

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