PinX1基因对鼻咽癌细胞顺铂化疗敏感性的影响及其机制

Influence and mechanism of PinX1 gene on the chemotherapy sensitivity of nasopharyngeal carcinoma cells in response to Cisplatin

目的 探讨PinX1基因对鼻咽癌细胞顺铂化疗敏感性的影响及其机制.方法 采用慢病毒构建的pCDH-CMV-PinX1-copGFP质粒载体转染鼻咽癌细胞,构建含PinX1基因载体的鼻咽癌Lenti-PinX1-5-8F细胞,同时构建Lenti-Ctrl-5-8F细胞（将不含PinX1基因的空载体转染5-8F细胞获得）及5-8F细胞作对照.将上述鼻咽癌细胞依实验分为4组：实验组为Lenti-PinX1-5-8F细胞＋顺铂组（含PinX1基因的Lenti-PinX1-5-8F细胞中加入顺铂药物）,对照组为Lenti-PinX1-5-8F细胞组（含PinX1基因）、顺铂药物组（5-8F细胞中加入顺铂）及5-8F细胞组.分别采用荧光定量PCR、流式细胞术、四甲基偶氮唑蓝（MTT）比色法、小室技术、Western印迹法及药物敏感试验检测PinX1基因表达、端粒酶活性、鼻咽癌增殖抑制、与顺铂抗癌的协同作用及肺耐药相关蛋白（LRP）及B细胞淋巴瘤基因-2（Bcl-2）基因的表达,观察PinX1基因在鼻咽癌细胞中对顺铂化疗敏感性的影响及其机制.结果 Lenti-PinX1-5-8F细胞中端粒酶活性相对量均显著低于Lenti-Ctrl-5-8F细胞、5-8F细胞（0.146±0.004比0.967 ±0.016、1.000±0.034,均P＜0.01）.16 μg/ml的顺铂与PinX1基因联合能显著增强端粒酶活性下调后对鼻咽癌细胞的抑制作用.Lenti-PinX1-5-8F细胞＋顺铂组的细胞增殖指数均显著低于Lenti-PinX1-5-8F细胞组、顺铂药物组、5-8F细胞组[（14.39±3.66）％比（32.97±3.00）％、（31.18±4.24）％、（47.19±4.19）％,均P＜0.01].Lenti-PinX1-5-8F细胞中LRP、Bcl-2蛋白相对表达量分别为0.64±0.14、0.57 ±0.12,均显著低于Lenti-Ctrl-5-8F细胞的0.84±0.19、0.81±0.16和5-8F细胞的0.83±0.35、0.78±0.27（均P＜0.01）.结论 PinX1基因能增强顺铂对鼻咽癌细胞的化疗敏感性,其作用有可能是通过下调鼻咽癌细胞端粒酶活性、抑制Bcl-2和LRP基因而实现的.

Objective To explore the influence and mechanism of PinX1 gene on the chemotherapy sensitivity of nasopharyngeal carcinoma cells in response to Cisplatin.Methods Transfeeted nasopharyngeal carcinoma 5-8F cell lines with pCDH-CMV-PinX1-copGFP vector constructed by lentivirus to generate LentiPinX1-5-8F cells containing PinX1 gene,using Lenti-Ctrl-5-8F cell （blank vector without PinX1 gene was used to transfect 5-8F cell lines） and 5-8F cell as controls.Expression of PinX1 gene,telomerase activity,the inhibition of cancer cells proliferation,combined anticancer effect with Cisplatin and the expression of lung resistance protein （LRP） and Bcl-2 were detected with fluorescent quantitation polymerase chain reaction （PCR）,flow cytometry,thiazolyl blue （MTT） method,areole test,Western blot and drug sensitivity test,respectively,in four groups （Lenti-PinX1-5-8F cell ＋ Cisplatin,Lenti-PinX1-5-8F cell,Cisplatin and 5-8F cell） so as to explore the influence and mechanism of PinX1 gene on the chemotherapy sensitivity of nasopharyngeal carcinoma cells in response to Cisplatin.Results The telomerase activity in Lenti-PinX1-5-8F cell （0.146 ± 0.004） was lower than those in the other two control cells （Lenti-Ctrl-5-8F cell：0.967 ±0.016,5-8F cell：1.000 ± 0.034,both P 〈 0.01）.The cancer cell biological activity could be intensively inhibited by 16 μg/ml Cisplatin after lower level telomerase activity induced by PinX1 gene.Proliferation index （PI） （%） in Lenti-PinXl-5-8F cell ＋ Cisplatin （14.39 ±3.66） was also less than the other groups （Lenti-PinX1-5-8F cell,Cisplatin and 5-8F cell groups,32.97 ± 3.00,31.18 ± 4.24 and 47.19 ±4.19,all P 〈0.01）.And same time,the expressions of LRP （0.64 ±0.14） and Bcl-2 （0.57 ± 0.12） protein in Lenti-PinX1-5-8F cells were obviously reduced than those in other two group cells （LentiCtrl-5-8F cell：0.84±0.19 and 0.81 ±0.16;5-8F cell：0.83±0.35 and 0.78±0.27;all P 〈0.01）.Conclusions PinX1 gene can enhance the chemotherapy sensitivity of nasopharyngeal carcinoma cells in response to Cisplatin,which may be mediated by the down-regulation of telomerase activity and the inhibition of LRP and Bcl-2 gene in nasopharyngeal carcinoma cells.