摘要
目的:探讨盐酸氯丙嗪对膀胱癌BT-B细胞迁移功能的影响及其机制。方法:用不同浓度的氯丙嗪处理膀胱癌BT-B细胞,通过MTT、细胞划痕和Transwell迁移实验验证氯丙嗪对细胞生长和迁移功能的影响。蛋白印迹实验和RT-qPCR实验验证氯丙嗪对Yes相关蛋白1(YAP1)和Ras相关蛋白1A(RAP1A)蛋白表达水平和mRNA表达水平的影响。免疫荧光实验验证氯丙嗪对YAP1细胞亚定位的影响。质粒转染实验验证YAP1和RAP1A与上皮间质转化(EMT)的调控关系。结果:氯丙嗪可抑制膀胱癌BT-B细胞的生长(t=11.53,P<0.001;t=35.83,P<0.0001;t=36.1,P<0.0001;t=194.6,P<0.0001)和迁移(t=5.80,P<0.01;t=7.43,P<0.01;t=5.13,P<0.01;t=6.61,P<0.01;t=18.51,P<0.0001;t=19.04,P<0.0001);与对照组相比,氯丙嗪作用于BT-B细胞后,YAP1(t=6.12,P<0.05;t=7.64,P<0.05)和RAP1A(t=4.87,P<0.05;t=8.30,P<0.05)的蛋白表达水平和mRNA表达水平(t=21.13,P<0.001;t=40.59,P<0.001)均显著降低;与对照组相比,氯丙嗪给药处理2 h后YAP1的荧光表达明显变弱;与对照组相比,YAP1表达增加后RAP1A蛋白(t=9.05,P<0.05)和EMT相关分子N-cadherin蛋白、Slug+snail蛋白(t=7.24,P<0.05;t=5.57,P<0.05)表达均显著增加,而YAP1敲低后RAP1A蛋白(t=4.36,P<0.05)、N-cadherin蛋白和Slug+snail蛋白(t=4.50,P<0.05;t=4.49,P<0.05)表达均显著降低。结论:氯丙嗪可通过YAP1调节RAP1A抑制膀胱癌的迁移,可能是治疗膀胱癌的一种新选择。
Objective:To investigate the effect and mechanism of chlorpromazine(CPZ)on the migration of bladder cancer BT-B cells.Methods:The MTT,wound healing and Transwell migration assays were used to determine the growth and migration of BT-B cells with different concentrations of CPZ treatment.Western blotting and RT-qPCR assays were used to determine the protein and mRNA expressions of Yes-associated protein 1(YAP1)and Ras-associated protein 1A(RAP1A).Immunofluorescence assay was used to verify the effect of CPZ on the sublocalization of YAP1.Plasmid transfection experiment was used to verify the regulatory relationship between YAP1 and RAP1A and epithelial-mesenchymal transition(EMT).Results:CPZ inhibited the growth(t=11.53,P<0.001;t=35.83,P<0.0001;t=36.1,P<0.0001;t=194.6,P<0.0001)and migration of bladder cancer BT-B cells(t=5.80,P<0.01;t=7.43,P<0.01;t=5.13,P<0.01;t=6.61,P<0.01;t=18.51,P<0.0001;t=19.04,P<0.0001).Compared with the control group,the expression of YAP1(t=6.12,P<0.05;t=7.64,P<0.05)and RAP1A(t=4.87,P<0.05;t=8.30,P<0.05),and the mRNA expression levels(t=21.13,P<0.001;t=40.59,P<0.001)of them were significantly decreased after treated with CPZ.Compared with the control group,the fluorescent expression of YAP1 was weaker with CPZ treatment at 2 hours.Compared with the control group,overexpression of YAP1 significantly increased the protein expressions of RAP1A(t=9.05,P<0.05),EMT-related molecules N-cadherin and Slug+snail(t=7.24,P<0.05;t=5.57,P<0.05),while the expression of RAP1A protein(t=4.36,P<0.05),N-cadherin protein and Slug+snail protein(t=4.50,P<0.05;t=4.49,P<0.05)were significantly decreased after the knockdown of YAP1.Conclusion:CPZ can inhibit the migration of bladder cancer by regulating RAP1A through YAP1.CPZ may be a new option for treating bladder cancer.
作者
陆益
于佳熙
梁政
LU Yi;YU Jia-xi;LIANG Zheng(Graduate School,Tianjin Medical University,Tianjin 300070,China;Department of Otorhinolaryngology,General Hospital,Tianjin Medical University,Tianjin 300052,China)
出处
《天津医科大学学报》
2022年第6期621-626,共6页
Journal of Tianjin Medical University
基金
天津市自然科学基金(17JCYBJC25600)。
