端粒酶抑制剂抑制鼠类肉瘤病毒癌基因突变肺癌细胞的恶性表型

摘要

目的 观察端粒酶抑制剂(BIBR1532)对含有鼠类肉瘤病毒癌基因(KRAS)突变肺癌细胞的影响.方法 利用慢病毒载体构建稳定表达KRAS第12外显子突变(所编码的氨基酸由甘氨酸突变为天冬氨酸,即G12D突变)的肺癌细胞株.Western blot检测G12D的表达水平.实时定量聚合酶链反应(Real-time PCR)法检测端粒酶活性.噻唑蓝(MTTT)法、划痕实验检测端粒酶抑制剂对表达G12D的肺癌细胞增殖、药物敏感性和迁移的影响.结果 成功建立了稳定表达KRAS-G12D的CALU3和H1299细胞株;BIBR1532抑制了过表达KRAS-G12D的CALU3和H1299细胞端粒酶活性(抑制率约为50％);BIBR1532对KRAS-G12D诱导的促肺癌细胞增殖能力、促肺癌细胞迁移能力和化疗抗性(细胞对顺铂和紫杉醇的敏感性提高了10％ ～15％和5％～12％)都产生了明显抑制.结论 端粒酶抑制剂能够抑制KRAS-G12D突变肺癌细胞的增殖、迁移,提高KRAS-G12D突变肺癌细胞对化疗药物的敏感性.%Objective To explore the effects of telomerase inhibitor BIBR1532 on lung cancer cells with kirsten rat sarcoma viral oncogene (KRAS) mutation.Methods Human lung cancer cells (CALU3/H1299) stably transfected with lentiviral vector-mediated KRAS-mutant (the encoded amino acid from glycine mutated to aspartic acid,G12D) expression were established and the telomerase activity and the expression levels of G12D were respectively detected by real-time quantitative polymerase chain reaction (Real-time PCR) and Western blotting.The proliferative activity,chemotherapy sensitivity and metastatic capability of cells were examined by methyl thiazol tetrazolium (MTI) and Scratch test.Results The telomerase activity level in KRAS-mutant lung cancer cells with telomerase inhibitors was more significantly decreased compared to KRAS wild type (KRAS-WT) lung cancer cells(the inhibition rates were about 50％).Relative to KRAS-WT cells,the KRAS-mutant cells with BIBR1532 grew and migrated more slowly,moreover,the inhibition ratio was improved more greatly (cisplatin and taxol respectively increased 10％-15％ and 5％-12％).Conclusion The telomerase inhibitors could inhibit proliferative ability and metastatic capability,and increase chemotherapy sensitivity of KRAS-mutant and KRAS-WT lung cancer cells,but the variation of KRAS-mutant lung cancer cells was greater.