口腔鳞癌对顺铂耐药分子机制探讨

Molecular mechanism of oral squamous cell carcinoma resistance to cisplatin

目的 Muc16是细胞表面黏蛋白,在多种肿瘤中过表达,Muc16C端可结合β-链蛋白(β-catenin)入核调节原癌基因c-Myc转录,进而调节卵巢癌细胞生长。本研究建立耐顺铂口腔鳞癌模型细胞,探讨Muc16-c-Myc信号通路对其调节作用,为临床治疗口腔鳞癌提供新思路。方法采用逐步增加顺铂(cis-Dichlorodiamineplatinum,DDP)药物浓度、间歇诱导人口腔鳞癌Tca8113细胞的方法建立口腔鳞癌体外耐药细胞模型(Tca8113/CDDP);MTT法检测药物对细胞的抑制率并计算半数抑制浓度(half maximal inhibitory concentration,IC50);siRNA转染沉默c-Myc蛋白表达,设立沉默组(c-Myc si)和空载对照组(c-Myc NC);Sh-RNA-Muc16转染细胞以沉默Muc16蛋白表达,设立沉默组(Muc16sh)和空载对照组(Muc16NC),并以嘌呤霉素筛选稳定表达细胞株;应用蛋白质印迹法检测各蛋白表达水平;结晶紫染色观察DDP处理后各组细胞存活率。结果与Tca8113细胞相比,Tca8113/CDDP中多药耐药相关蛋白1(multidrug resistanec-associated protein 1,MRP1)上调(44.5±7.3)%,z=-2.087,P<0.001;P-糖蛋白(P-glycoprotein,P-gp)表达上调(30.1±7.7)%,z=-2.016,P<0.001。Muc16沉默后,与Muc16NC组相比,Muc16sh组Tca8113/CDDP中Muc16表达下调(91.8±3.8)%,z=-5.087,P<0.001;与Muc16 NC组相比,Muc16sh组细胞c-Myc表达下调(42.3±5.2)%,z=-4.165,P<0.001。Muc16沉默后Tca8113/CDDP中MRP1表达下调(67.7±6.2)%,z=-3.656,P<0.001;P-gp表达下调(59.6±8.1)%,z=-3.884,P<0.001。Tca8113/CDDP中c-Myc沉默后,其蛋白表达量下降(78.8±4.8)%,z=-5.112,P<0.001;c-Myc沉默后,Tca8113/CDDP细胞MRP1表达下调(53.2±3.2)%,z=-5.745,P<0.001;P-gp表达下调(74.6±7.7)%,z=-3.948,P<0.001。Muc16 NC组DDP的IC50为(165.4±8.5)μg/mL,Muc16sh组为(72.9±4.1)μg/mL,与Muc16NC组相比,Muc16sh组IC50明显降低,z=-3.312,P<0.001;c-Myc NC组DDP的IC50为(157.2±6.3)μg/mL,c-Myc si组IC50为(58.3±3.8)μg/mL,与c-Myc NC组相比,c-Myc si组细胞IC50明显降低,z=-4.606,P<0.001。DDP(165.4μg/mL)处理细胞24h发现,与Muc16NC组相比,Muc16sh组Tca8113/CDDP存活率下降(70.1±8.1)%,z=-2.787,P<0.001;DDP(157.2μg/mL)处理细胞24h发现,与c-Myc NC组相比,c-Myc si组Tca8113/CDDP存活率下降(67.6±7.7)%,z=-3.568,P<0.001。结论Tca8113/CDDP通过活化Muc16-c-Myc信号通路维持细胞耐药蛋白MRP1和P-gp的表达,从而对DDP耐药。

OBJECTIVE The membrane-associated mucin Muc16,a heavily O-glycosylated transmembrane protein,localizes on the cell surface and is overexpressed in several tumors.The C-terminal domain of Muc16 can bind toβ-catenin,through which Muc16 transports into the nucleus,and then regulates the growth of ovarian cancer cells.The aim of this study was to establish a model of cisplatin（DDP）-resistant oral squamous cell carcinoma cell line,and to explore the regulation effect of Muc16-c-Myc signaling pathway in DDP-resistant cells,providing a new way for treatment of oral squamous cell carcinoma in clinic.METHODS The DDP-resistant oral squamous cell carcinoma cell line（Tca8113/CDDP）was established by intermittent exposure to gradually increasing doses of DDP.The inhibitory effect of drug on the cells was detected by tetramethylzole（MTT）method.Transfection of siRNA was used to silence c-Myc expression,and transfected cells were separated into silent group（Muc16 sh）and empty plasmid control group（Muc16 NC）,respectively.In addition,shRNA-MUC16 was transfected into the cells to silence Muc16 expression,and puromycin selection was applied for screening cell lines with stable protein expression.The western blot method was used to detect the expression levels of proteins,and cell viability was observed by crystal violet staining.RESULTS As compared with Tca8113 cells,the expression levels of multidrug resistance-related protein1（MRP1）and P-glycoprotein（P-gp）in Tca8113/CDDP cells were up-regulated by（44.5±7.3）%（z=-2.087,P〈0.001）and（30.1±7.7）%（z=-2.016,P〈0.001）,respectively.As compared with Muc16 NC group,the expression level of Muc16 of Tca8113/CDDP in Muc16 sh group was downregulated by（91.8±3.8）% after Muc16 silencing,z=-5.087,P〈0.001,and the expression of c-Myc in Muc16 sh group was down-regulated by（42.3±5.2%）,z=-4.165,P〈0.001.The expression levels of MRP1 and P-gp in Tca8113/CDDP cells after MUC16 silencing were decreased by（67.7±6.2）%（z=-3.656,P〈0.001）and（59.6±8.1）%（z=-3.884,P〈0.001）,respectively.Silencing of c-Myc in Tca8113/CDDP cells led to a decrease in c-Myc expression by（78.88±4.83）%,z=-5.112,P〈0.001.After c-Myc silencing,the expressions of MRP1 and P-gp were decreased by（53.2±3.2）%（z=-5.745,P〈0.001）and（74.6±7.7）%（z=-3.948,P〈0.001）,respectively.The50%inhibitory concentration（IC50）of DDP was（165.4±8.5）μg/mL in Muc16 NC group,while IC50 value was（72.9±4.1）μg/mL in Muc16 sh group,which was significantly lower than that of Muc16 NC group,z=-3.312,P〈0.001.The IC50 values of DDP were（157.2±6.3）μg/mL and（58.3±3.8）μg/mL in c-Myc NC and c-Myc si groups,respectively.As compared with c-Myc NC group,IC50 was notably decreased,z=-4.606,P〈0.001.24 hafter exposure to DDP（165.4μg/mL）,cell viability of Tca8113/CDDP in Muc16 sh goup was decreased by（70.1±8.1）%,z=-2.787,P〈0.001;24 hafter exposure to lower dose DDP（157.2μg/mL）,cell viability of Tca8113/CDDP in c-Myc si group was decreased by（67.6±7.7）%,z=-3.568,P〈0.001.CONCLUSION CDDP-resistant oral squamous cell carcinoma cell Tac8113 would maintain the expression of drug-resistant proteins MRP1 and P-gp by regulation of the Muc16-c-Myc signaling pathway,which may led to its resistance to DDP.