二甲双胍通过干预上皮-间质转化逆转尼古丁诱导的 肺癌吉非替尼耐药

Metformin reverses nicotine-induced gefitinib resistance by epithelial-mesenchymal transition

摘要:

目的：探讨尼古丁（nicotine，NIC）诱导EGFR敏感突变PC-9肺癌细胞株吉非替尼耐药及二甲双胍对其作用。方法：PC-9分成4组：空白组、尼古丁组、二甲双胍组、尼古丁和二甲双胍组，利用定量反转录聚合酶连锁反应（quantificational real-time polymerase chain reaction，qRT-PCR）检测E-cadherin和Vimentin信使核糖核酸（messenger ribonucleic acid，mRNA）的表达，Western blot检测E-cadherin和Vimentin蛋白表达，细胞计数试剂盒（cell counting Kit-8，CCK-8）检测不同分组细胞对吉非替尼药物敏感情况。Transwell小室实验检测细胞侵袭迁移。结果：尼古丁诱导PC-9细胞上皮间质转化呈时间浓度依赖，在10 μmol/L尼古丁处理72 h后，发生E-cadherin下调和Vimentin上调最明显，故选该条件做以下实验。CCK-8提示，相比空白组，尼古丁组PC-9细胞对吉非替尼敏感性减弱，在吉非替尼浓度为0.05 μmol/L最明显（P=0.000）。Matrigel侵袭实验提示尼古丁组穿膜细胞数较空白组明显增多[（15.70±1.53）个/高倍视野 vs. （32.70±2.08）个/高倍视野，F=75.406，P=0.000，P=0.000]；迁移实验的结果与之相似。与空白组相比，尼古丁组E-cadherin mRNA 表达水平明显降低[（0.932±0.100） vs. （0.459±0.024），F=25.924，P=0.000，P=0.000）]，Vimentin mRNA表达水平明显升高[（1.200±0.200） vs. （1.973±0.129），F=20.998，P=0.000，P=0.000]。蛋白水平变化与之相同。尼古丁＋二甲双胍组可以增加尼古丁作用PC-9对吉非替尼的敏感性，在吉非替尼浓度为0.05、0.10、0.50、1.00、5.00 μmol/L时有统计学意义。尼古丁＋二甲双胍组可以减弱尼古丁组的穿膜数[（17.00±2.00）个/高倍视野vs. （32.70±2.08）个/高倍视野，F=75.406，P=0.000，P=0.000]，迁移实验呈现相同的结果。相比于尼古丁组，尼古丁＋二甲双胍组上调E-cadherin mRNA[（0.459±0.024） vs. （0.838±0.058），F=25.924，P=0.000，P=0.000）]，下调Vimentin mRNA[（1.973±0.129） vs. （1.467±0.115），F=20.998，P=0.000，P=0.003）]。结论：尼古丁通过上皮间质转化诱导PC-9细胞吉非替尼耐药，二甲双胍可以逆转上述现象。

Abstract:

Objective：To investigate the molecular basis for nicotine（NIC） to induce resistance of epithelial growth factor receptor tyrosine kinase inhibitors（EGFR-TKI） in EGFR-mutated PC-9 cells and the effect of the metformin in the process. Methods：PC-9 cells were divided into 4 groups，namely control group，NIC group，MET group and MET+NIC group. EMT markers were detected by quantificational real-time polymerase chain reaction（qRT-PCR） and Western blot. Cell counting Kit-8（CCK-8） assays were used to evaluate the sensibility to gefitinib of different group cells. The invasion ability and migration ability of different groups were detected by Transwell assay. Results：Nicotine in－duced epithelial-mesenchymal transition（EMT） of PC-9 cells in a time and concentration dependent manner. Down-regulation of E-cadherin and up-regulation of Vimentin were most significant with a treatment of 10 μmol/L nicotine for 10 h，so further experi－ments were completed under this condition. CCK-8 displayed reduction of gefitinib sensitivity of PC-9 cells in the NIC group com－pared with the control group，especially with a gefitinib concentration of 0.05 μmol/L（F=82.005，P=0.000，P=0.000）. In invasion as－says，more penetrating cells were observed in the NIC group than in the control group[（15.70±1.53） vs. （32.70±2.08），F=75.406，P=0.000，P=0.000]. Migration assays presented similar results [（102.70±7.02） vs. （18.70±2.08），F=204.038，P=0.000，P=0.000]. E-cadherin mRNA expression decreased[（0.932±0.100） vs. （0.459±0.024），F=25.924，P=0.000，P=0.000] and Vimentin mRNA expression increased[（1.200±0.200） vs. （1.973±0.129），F=20.998，P=0.000，P=0.000] in the NIC group compared with the control group，and Western blot showed the same results. Cells in the MET+NIC group displayed higher sensitivity to gifitinib，which is statis－tically significant with a gifitinib concentration of 0.05，0.10，0.50，1.00 and 5.00 μmol/L. Fewer cells penetrated the Matrigel in the NIC+MET group[（17.00±2.00） vs. （32.70±2.08），F=75.406，P=0.000，P=0.000] and migration assays presented similar results [（51.70±5.03） vs. （102.70±7.02），F=204.038，P=0.000，P=0.000]. E-cadherin mRNA expression was upregulated[（0.459±0.024） vs. （0.838±0.058），F=25.924，P=0.000，P=0.000]，and Vimentin mRNA expression was downregulated [（1.973±0.129） vs. （1.467±0.115），F=20.998，P=0.000，P=0.003] in the NIC+MET group compared with the NIC group. Western blot showed the same results. Conclusion：Nicotine induced EGFR-TKI resistance in PC-9 cells by EMT in non-small cell lung cancer，which could be reversed by metformin.