同源盒基因2在黑色素瘤细胞对于曲美替尼耐药中的作用

doi:10.12092/j.issn.1009-2501.2019.10.010

摘要/Abstract

摘要：

目的:研究同源盒基因2（MSX2）在黑色素瘤细胞A375对于曲美替尼耐药中的作用机制。方法:构建曲美替尼耐药的A375细胞株（A375-AR），曲美替尼干预A375和A375-AR后采用CCK-8检测细胞活力，流式细胞术检测细胞凋亡率，蛋白免疫印迹法（Western blot）检测细胞中Bcl-2、Bax、Caspase-3、Caspase-9的表达以及MSX2的表达。采用小干扰RNA（siRNA）沉默A375-AR中MSX2基因，设计A375、A375-AR、A375-AR-MSX2，曲美替尼干预后，CCK-8检测细胞活力，流式细胞术检测细胞凋亡率，Western blot检测细胞中Bcl-2、Bax、Caspase-3、Caspase-9的表达以及MSX2的表达。构建MSX2过表达的A375细胞系（A375-OE），设置A375、A375-OE和A375-AR组，曲美替尼干预后，CCK-8检测细胞活力，流式细胞术检测细胞凋亡率，Western blot检测细胞中Bcl-2、Bax、Caspase-3、Caspase-9的表达以及MSX2的表达。结果:A375-AR对1.8 nmol/L的曲美替尼耐药，采用1.8 nmol/L曲美替尼干预后，A375细胞活力和细胞凋亡率显著高于A375-AR，A375细胞中Bcl-2基因表达水平低于A375-AR，而Bax、Caspase-3、Caspase-9的表达水平高于A375-AR。A375-AR沉默MSX2后，细胞对于曲美替尼敏感性显著增高，且细胞活力下调，凋亡率上调，细胞中Bcl-2表达下调，Bax、Caspase-3、Caspase-9的表达上调。A375过表达MSX2后，细胞对于曲美替尼敏感性显著下调，细胞活力上调，凋亡率下调，细胞中Bcl-2表达上调，Bax、Caspase-3、Caspase-9的表达下调。结论:MSX2基因可以诱导黑色素瘤细胞对于曲美替尼的耐药，是治疗黑色素瘤耐药的潜在靶点。

关键词: 同源盒基因MSX2, 黑色素瘤, 曲美替尼, 耐药

Abstract:

AIM: To study the role of homeobox gene MSX2 in the melanoma cell line A375 of trametinib resistance. METHODS: To construct trametinib resistant A375 cell line (A375-AR). After trametinib intervented A375 and A375-AR，the cell viability was detected by CCK-8, the cell apoptosis rate was detected by flow cytometry, and the expression of Bcl-2, Bax, Caspase-3, Caspase-9 and MSX2 in the cells were detected by Western blot. Small interfering RNA (siRNA) was used to silence MSX2 gene in A375-AR. After trametinib intervented A375 and A375-AR，the cell viability was detected by CCK-8, the cell apoptosis rate was detected by flow cytometry, and the expression of Bcl-2, Bax, Caspase-3, Caspase-9 and MSX2 in the cells were detected by Western blot. To construct MSX2 over-expressing A375 cell line (A375-OE), and A375, A375-OE and A375-AR groups were set up. After trametinib intervented A375 and A375-AR，the cell viability was detected by CCK-8, the cell apoptosis rate detected by flow cytometry, and the expression of Bcl-2, Bax, Caspase-3, Caspase-9 and MSX2 in the cells were detected by Western blot. RESULTS:A375-AR was resistant to trametinib of 1.8 nmol/L, the activity of A375 cells and the rate of cell apoptosis were significantly higher than that of A375-AR, and the expression level of Bcl-2 gene in A375 cells was lower than that of A375-AR, while the expression level of Bax, Caspase-3 and Caspase-9 was higher than that of A375-AR. After A375-AR silenced of MSX2, the cells were significantly increased to the sensibility of trametinib, the apoptosis rate was up-regulated, the expression of Bcl-2 in the cells was down-regulated, and the expression of Bax, Caspase-3 and Caspase-9 was up-regulated. After A375 overexpression of MSX2, the cells were down regulated to the sensibility of trametinib, the cell viability was up-regulated, the apoptosis rate was down-regulated, the expression of Bcl-2 in the cells was up-regulated, and the expression of Bax, Caspase-3 and Caspase-9 was down regulated. CONCLUSION: MSX2 gene can induce the resistance of melanoma cells to trametinib. It is a potential target for the treatment of melanoma resistance.

Key words: homeobox gene MSX2, melanoma, trametinib, drug resistance

中图分类号:

R966
R739.5

王小丽，陆树萍，戴加乐. 同源盒基因2在黑色素瘤细胞对于曲美替尼耐药中的作用[J]. 中国临床药理学与治疗学, 2019, 24(10): 1147-1154.

WANG Xiaoli, LU Shuping, DAI Jiale. Role of MSX2 in melanoma cells for trametinib resistance[J]. Chinese Journal of Clinical Pharmacology and Therapeutics, 2019, 24(10): 1147-1154.

参考文献

［1］Andrade MC, Carmo LS, Fariassilva E, et al. Msx2 is required for vascular smooth muscle cells osteoblastic differentiation but not calcification in insulin-resistant ob/ob mice［J］. Atherosclerosis, 2017, 265:14-21.
［2］Amri N, Djolé SX, Petit S, et al. Distorted patterns of dentinogenesis and eruption in Msx2 null mutants: involvement of Sost/Sclerostin［J］. Am J Pathol, 2016, 186(10):2577-2587.
［3］Liang H, Zhang Q, Lu J, et al. MSX2 induces trophoblast invasion in human placenta:［J］. Plos One, 2016, 11(4):e0153656.
［4］Satoh K, Hamada S, Shimosegawa T. Pancreatic tumor and epithelial to mesenchymal transition (EMT) -The role of MSX2 in EMT of pancreatic tumor cells-［J］. Suizo, 2014, 29(1):13-22.
［5］Burke S, Lakshmikanth T, Colucci F, et al. New views on natural killer cell-based immunotherapy for melanoma treatment［J］. Trends Immunol, 2010, 31(9):339-345.
［6］Aris M, Barrio MM. Combining immunotherapy with oncogene-targeted therapy: a new road for melanoma treatment［J］. Front Immunol, 2015, 6:46.
［7］Boussemart L. Improved overall survival in melanoma with combined dabrafenib and trametinib［J］. N Engl J Med, 2015, 372(1):30-39.
［8］Sun J, Ting M C, Ishii M, et al. Msx1 and Msx2 function together in the regulation of primordial germ cell migration in the mouse［J］. Dev Biol, 2016, 417(1):11-24.
［9］Taghiyar L, Hesaraki M, Sayahpour FA, et al. Msh homeobox 1 (Msx1)- and Msx2-overexpressing bone marrow-derived mesenchymal stem cells resemble blastema cells and enhance regeneration in mice［J］. J Biol Chem, 2017, 292(25): 10520-10533.
［10］Hamada S, Satoh K, Hirota M, et al. Bone morphogenetic protein 4 induces epithelial-mesenchymal transition through MSX2 induction on pancreatic cancer cell line［J］. J Cell Physiol, 2010, 213(3):768-774.
［11］Kennichi S, Shin H, Kenji K, et al. Up-regulation of MSX2 enhances the malignant phenotype and is associated with twist 1 expression in human pancreatic cancer cells ［J］. Am J Pathol, 2008,172(4):926.
［12］Satoh K, Hamada S, Kanno A, et al. Msx-2 enhances the malignant phenotype of human pancreatic cancer cells, in vitro and in vivo［J］. Cancer Res, 2006, 66(2):852-863.
［13］宋瑞鹏, 杨海彦, 赵宝磊,等. MSX2基因与上皮间质化在肿瘤浸润转移的研究进展［J］. 中华实验外科杂志, 2010, 27(4):543-544.
［14］Lanigan F, Gremel G, Hughes R, et al. Homeobox transcription factor muscle segment homeobox 2 (Msx2) correlates with good prognosis in breast cancer patients and induces apoptosis in vitro［J］. Breast Cancer Res, 2010, 12(4):R59.
［15］Jr IM, Wang W, Young AT. Fibroblast growth factors lead to increased Msx2 expression and fusion in calvarial sutures［J］. J Bone Miner Res, 2010, 18(4):751-759.

[1]	姜道利, 丑晓华, 刘志东, 李薇, 张波, 许桑, 谈德斐, 方翼, 吕冬梅, 王涛. 头孢他啶阿维巴坦治疗多重耐药革兰阴性菌感染的真实世界研究[J]. 中国临床药理学与治疗学, 2023, 28(9): 1008-1017.
[2]	何李华, 朱秀之, 江一舟. 三阴性乳腺癌免疫治疗研究进展[J]. 中国临床药理学与治疗学, 2023, 28(8): 842-853.
[3]	张胡云龙, 朱秀之, 金希, 邵志敏. 腔面型乳腺癌内分泌治疗耐药的机制与治疗进展[J]. 中国临床药理学与治疗学, 2023, 28(8): 854-865.
[4]	李珊, 柳熠鑫, 任菲菲, 李相晨, 张志清. 天然植物活性成分通过下调 P-糖蛋白逆转肿瘤多药耐药的研究进展[J]. 中国临床药理学与治疗学, 2023, 28(3): 331-340.
[5]	刘春, 王萌, 程卉, 李庆林. 基于RNA-seq测序技术研究新藤黄酸抑制黑色素瘤增殖的作用机制[J]. 中国临床药理学与治疗学, 2023, 28(2): 155-163.
[6]	王秀, 季麒麟, 裴文俊, 曾颖. 3-溴丙酮酸-胆固醇酯增强乳腺癌细胞对他莫昔芬敏感性研究[J]. 中国临床药理学与治疗学, 2023, 28(10): 1093-1100.
[7]	黄好, 贾虹, 王晓霜, 张鹭, 蒋华. 负载MAGE-A3抗原的树突状细胞与细胞因子诱导杀伤细胞共培养对子宫内膜癌肿瘤干细胞及恶性进展的影响[J]. 中国临床药理学与治疗学, 2023, 28(1): 42-50.
[8]	刘佳佳, 高岩, 张业慧, 武静. CRABP2在肿瘤发生发展中的作用研究[J]. 中国临床药理学与治疗学, 2023, 28(1): 86-92.
[9]	周欢, 徐铭, 李波, 刘春英, 王淳. PI3K/Akt/FOXO3a信号通路介导的保护性自噬参与肺腺癌获得性顺铂耐药表型重塑[J]. 中国临床药理学与治疗学, 2022, 27(9): 961-970.
[10]	张可心, 贾文静, 崔佳文, 敖路遥, 周芳, 王广基, 刘嘉莉. 第三代EGFR酪氨酸激酶抑制剂在非小细胞肺癌临床治疗中的应用及研究进展[J]. 中国临床药理学与治疗学, 2022, 27(9): 1016-1030.
[11]	董伟, 黄小英, 谢冰斌, 汤喜兰, 李洪铭, 邱雨美, 赵国巍, 梁新丽. 氧化前胡素对乳腺癌MCF-7/DOX细胞多柔比星耐药的抑制作用[J]. 中国临床药理学与治疗学, 2022, 27(3): 260-266.
[12]	李玮, 胡佳丽, 王凯, 贺毅憬. 黑色素瘤免疫治疗的研究现状与展望[J]. 中国临床药理学与治疗学, 2021, 26(9): 1053-1064.
[13]	孟强, 刘克辛. 转运体介导药物相互作用的研究现状及展望[J]. 中国临床药理学与治疗学, 2021, 26(8): 876-888.
[14]	余赛红, 郑晓亮, 蒲依依, 颜冬梅, 王孝举, 于洁. 通过核糖核苷酸还原酶M2逆转肝癌细胞对5-氟尿嘧啶耐药[J]. 中国临床药理学与治疗学, 2021, 26(7): 729-737.
[15]	符祥俊, 黄莉, 郭丽, 林良沫. 万古霉素AUC/MIC在多重耐药革兰阳性菌致重症感染中的应用分析[J]. 中国临床药理学与治疗学, 2021, 26(7): 775-781.