O6-甲基鸟嘌呤-DNA甲基转移酶siRNA联合替莫唑胺对黑素瘤细胞增殖的影响
| 作者: |
1孙金中,
2黄倩,
2辛勇,
3蒋冠,
1孙圣荣
1 武汉大学人民医院乳腺甲状腺外科，武汉 430060 2 徐州医科大学附属医院放疗科，江苏 徐州 221002 3 徐州医科大学附属医院皮肤科，江苏 徐州 221002 |
| 通讯: |
辛勇
Email: 5721268@163.com |
| DOI: | 10.3978/j.issn.2095-6959.2020.07.002 |
摘要
目的：研究O6-甲基鸟嘌呤-DNA甲基转移酶(O6-methylguananine-DNA methyltransferase，MGMT)对人黑素瘤细胞耐替莫唑胺(temozolomide，TMZ)的影响及机制。方法：体外培养人黑素瘤A375细胞、M14细胞、MV3细胞，采用RT-qPCR及蛋白质印迹法检测细胞MGMT的表达量；CCK-8法检测细胞的增殖抑制率；蛋白质印迹法检测不同干扰序列对黑素瘤细胞MGMT的干扰效果；蛋白质印迹法检测细胞内PARP，DNA-PKcs和NF-κB蛋白表达。结果：RT-PCR及蛋白质印迹法结果显示不同黑素瘤细胞MGMT表达量不同，其中A375细胞MGMT表达量最高，MV3细胞的表达量最少；蛋白质印迹法结果显示转染MGMT siRNA后黑素瘤细胞MGMT表达量明显下降(P<0.05)；CCK-8结果显示TMZ对A375细胞及M14细胞的生长增殖有抑制作用，并且这种作用呈时间和剂量依赖性(P<0.05)；蛋白质印迹法结果可见MGMT siRNA联合TMZ处理组PARP，DNA-PKcs，NF-κB蛋白表达减弱(P<0.05)。结论：MGMT siRNA联合TMZ显著抑制黑素瘤A375和M14细胞增殖。
关键词：
替莫唑胺；黑素瘤；细胞耐药；O6-甲基鸟嘌呤-DNA甲基转移酶
Effect of MGMT siRNA combined with temozolomide on the proliferation of melanoma cells
CorrespondingAuthor: XIN Yong Email: 5721268@163.com
DOI: 10.3978/j.issn.2095-6959.2020.07.002
Abstract
Objective: To investigate the effect of O6-methylguananine-DNA methyltransferase (MGMT) on the melanoma cells to resist to temozolomide (TMZ). Methods: Human melanoma A375 cells, M14 cells, MV3 cells were cultured in vitro. The expression of MGMT in cells was detected by RT-qPCR and Western blotting. Temozolomide on cell proliferation was analyzed by the CCK-8 method. The interference effect of different interference sequences of MGMT on melanoma cell line was detected by Western blot. The expression of PARP, DNA-Pkcs and NF-κB was detected by Western blotting. Results: The results of RT-PCR and Western Blot showed that the expression of MGMT was different in different melanoma cells. Western blot results showed that the expression of MGMT in melanoma cells decreased after transfecting with MGMT siRNA (P<0.05); temozolomide inhibited the growth and proliferation of A375 cells and M14 cells significantly through CCK-8 assays, and this effect was time and dose-dependent (P<0.05). Western blot showed that the expression of PARP, NF-κB and DNA-PKcs protein was decreased (P<0.05). Conclusion: Temozolomide inhibits the proliferation of A375 cells and M14 cells, and MGMT siRNA combined with temozolomide significantly inhibits the proliferation of melanoma A375 cells and M14 cells.
Keywords：
temozolomide; melanoma; drug resistance; O6-methylguananine-DNA methyltransferase