布托啡诺调节FOXO3-FOXM1信号轴对骨肉瘤细胞生物活性和化疗药物耐药性的实验研究  

Experimental Studies Butorphanol on the Biological Activity and Chemotherapy Drug Resistance of Osteosarcoma Cells by Regulating the FOXO3-FOXM1 Signal Axis

作　　者：奥婷婷 姜祯珍 张熙 AO Tingting;JIANG Zhenzhen;ZHANG Xi(Department of Anesthesiology,Shiyan People’s Hospital,Hubei Shiyan 442000,China)

机构地区：[1]十堰市人民医院麻醉科,湖北十堰442000

出　　处：《现代检验医学杂志》2024年第6期37-42,66,共7页Journal of Modern Laboratory Medicine

基　　金：湖北省卫生健康委科研项目(WJ2023F090)。

摘　　要：目的探究布托啡诺(BUT)调节叉头框蛋白O3(FOXO3)-叉头框蛋白M1(FOXM1)信号轴对骨肉瘤细胞生物活性和化疗药物耐药性的影响。方法将2.0μmol/L顺铂(CDDP)处理的CDDP耐药MG-63细胞(MG-63/CDDP)分为对照组(MG-63/CDDP细胞用含0.05g/dl DMSO培养液处理)、BUT组(40μg/ml BUT处理MG-63/CDDP细胞)、JY-2组(用100μmol/L FOXO3-FOXM1抑制剂JY-2处理MG-63/CDDP细胞)和BUT+JY-2组(用40μg/ml BUT以及100μmol/L JY-2处理MG-63/CDDP细胞)。CCK8法检测MG-63/CDDP细胞活性;流式细胞术检测MG-63/CDDP细胞凋亡情况;Transwell法检测MG-63/CDDP细胞迁移、侵袭情况;Western blot检测自噬蛋白以及FOXO3-FOXM1信号通路相关蛋白表达。结果与MG-63细胞相比,MG-63/CDDP细胞IC50增加(20.56±2.52μmol/L vs 0.97±0.10μmol/L),差异具有统计学意义(q=19.017,P<0.05),筛选出较适浓度1μmol/L CDDP用于后续实验。与对照组相比,BUT组MG-63/CDDP细胞A值(0.43±0.05 vs 0.68±0.06),细胞迁移数量(63.63±7.58个vs114.56±10.57个)以及侵袭数量(43.38±4.58个vs 79.56±8.48个)、自噬相关蛋白Beclin1(0.31±0.05 vs 0.62±0.07)和微管相关蛋白轻链3(LC3)-II/I蛋白(0.51±0.08 vs 0.98±0.11)水平均下降(q=6.763~9.591,均P<0.05),凋亡率(28.57%±3.14%vs 8.67%±1.46%),FOXO3(0.72±0.08 vs 0.33±0.04),FOXM1(1.22±0.15 vs 0.70±0.08)蛋白水平均上升(q=14.077,10.681,7.493,均P<0.05),而JY-2组MG-63/CDDP细胞A值(0.99±0.13 vs0.68±0.06),细胞迁移数量(147.59±15.37个vs 114.56±10.57个)以及侵袭数量(111.83±12.58个vs 79.56±8.48个),Beclin1(0.94±0.11 vs 0.62±0.07),LC3-II/I蛋白(1.27±0.13 vs 0.98±0.11)水平均升高(q=4.171~6.012,均P<0.05),凋亡率(4.56%±0.86%vs 8.67%±1.46%),FOXO3(0.17±0.01 vs 0.33±0.04),FOXM1(0.46±0.03 vs 0.70±0.08)蛋白水平降低(q=5.941,9.505,6.881,均P<0.05),差异具有统计学意义。JY-2逆转了BUT对MG-63/CDDP细胞活性和化疗耐药性的有利影响。结论BUT可能通过激活FOXO3-FOXM1信号通路调节骨肉瘤细胞的细胞活性和CDDP耐药�Objective To investigate the impacts of butorphanol(BUT)on the biological activity and chemotherapy drug resistance of osteosarcoma cells by regulating the forkhead box protein O3(FOXO3)-forkhead box protein M1(FOXM1)signal axis.Methods CDDP resistant MG-63 cells(MG-63/CDDP)treated with 2.0μmol/L cisplatin(CDDP)were separated into control group(MG-63/CDDP cells were treated with 0.05%DMSO medium),BUT group(MG-63/CDDP cells were treated with 40μg/mL BUT),JY-2 group(MG-63/CDDP cells were treated with 100μmol/L FOXO3-FOXM1 inhibitor JY-2),and BUT+JY-2 group(MG-63/CDDP cells were treated with 40μg/ml BUT and 100μmol/L JY-2).CCK8 method was applied to detect MG-63/CDDP cell activity.Flow cytometry was used to detect apoptosis in MG-63/CDDP cells.The Transwell method was applied to detect the migration and invasion of MG-63/CDDP cells;Western blot was applied to detect the expression of autophagy proteins and proteins related to the FOXO3-FOXM1 signaling pathway.Results Compared with MG-63 cells,the IC50(20.56±2.52μmol/L) vs(0.97±0.10μmol/L)of MG-63/CDDP cells was increased,and the differences was statistically significant(q=19.017,P<0.05),and the optimal concentration of 1μmol/L CDDP was selected for subsequent experiments.Compared with the control group,the A value(0.43±0.05 vs 0.68±0.06),numbers of cell migration(63.63±7.58 vs 114.56±10.57)and invasion(43.38±4.58 vs 79.56±8.48),and the levels of autophagy-related protein Beclin1(0.31±0.05 vs 0.62±0.07)and microtubule-associated protein light chain 3(LC3)-II/I proteins(0.51±0.08 vs 0.98±0.11)in the BUT group were reduced(q=6.763~9.591,all P<0.05),the apoptosis rate(28.57%±3.14%vs 8.67%±1.46%),the levels of FOXO3(0.72±0.08 vs 0.33±0.04)and FOXM1(1.22±0.15 vs 0.70±0.08)proteins were increased(q=14.077,10.681,7.493,all P<0.05),however,in the JY-2 group,the A value(0.99±0.13 vs 0.68±0.06),numbers of cell migration(147.59±15.37 vs 114.56±10.57)and invasion(111.83±12.58 vs 79.56±8.48),and the levels of Beclin1(0.94±0.11 vs 0.62±0.07)and LC

关 键 词：布托啡诺 叉头框蛋白O3-叉头框蛋白M1信号通路 骨肉瘤 顺铂 

分 类 号：R738.1[医药卫生—肿瘤] R730.43[医药卫生—临床医学]