EML4-ALK-G1202R点突变质粒及其体外稳转细胞系构建
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摘要
目的融合PCR法构建棘皮动物微管相关蛋白样4-间变性淋巴瘤激酶(echinoderm microtubule associated protein like 4-anaplastic lymphoma kinase,EM L4-ALK)G1202R点突变载体,并构建体外克唑替尼耐药细胞模型。方法利用RT-PCR从H2228细胞中获得EML4-ALK融合基因,随后利用融合PCR技术构建EML4-ALK-G1202R点突变融合基因,酶切后定向连入真核表达质粒pEGFP-N1中,构建pEGFP-N1-EM L4-ALK-G1202R重组质粒。脂质体法将重组质粒转入A549细胞中,G418抗生素筛选构建EML4-ALK-G1202R稳定高表达细胞系。MTT法验证细胞模型对克唑替尼的耐受性。结果测序结果表明EML4-ALK-G1202R点突变正确,Real time-PCR和Western blot检测结果表明稳转细胞系构建成功,M TT结果表明克唑替尼耐药模型构建成功。结论成功构建了pEGFP-N1-EML4-ALK-G1202R真核表达载体,转染A549细胞后,可稳定表达并发挥克唑替尼耐药性,为进一步研究EML4-ALK点突变导致的细胞耐药机制奠定了基础。
Objective To construct the recombinant plasmid carrying EML4-ALK-G1202 R fusion gene and obtain crizotinib drug resistant cell model. Methods EML4-ALK fusion gene was obtained from H2228 cells by RT-PCR and the G1202 R point mutation was constructed by fusion PCR. Then EML4-ALK fusion gene was inserted into vector pEGFP-N1 to build the pEGFP-N1-EML4-ALK-G1202 R recombinant plasmid,which was transfected into A549 cells and the cell lines which stably expressed the EML4-ALK-G1202 R fusion gene were screened by G418. The tolerance of cell model to crizotinib was detected by MTT. Results The results showed that the EML4-ALK-G1202 R point mutation was correct,and the stable transformation cell line was verified by the real time-PCR or western blot. MTT results showed that the crizotinib drug resistant model was successfully constructed. Conclusion pEGFP-N1-EML4-ALK-G1202 R eukaryotic expression vector was successfully constructed,and EML4-ALK-G1202 R can be stably expressed after being transfected to A549 cell,and could develop the drug resistance of crizotinib. Our experiment laid a foundation for further mechanism study on EML4-ALK mutations-leaded cell resistance.
Objective To construct the recombinant plasmid carrying EML4-ALK-G1202 R fusion gene and obtain crizotinib drug resistant cell model. Methods EML4-ALK fusion gene was obtained from H2228 cells by RT-PCR and the G1202 R point mutation was constructed by fusion PCR. Then EML4-ALK fusion gene was inserted into vector pEGFP-N1 to build the pEGFP-N1-EML4-ALK-G1202 R recombinant plasmid,which was transfected into A549 cells and the cell lines which stably expressed the EML4-ALK-G1202 R fusion gene were screened by G418. The tolerance of cell model to crizotinib was detected by MTT. Results The results showed that the EML4-ALK-G1202 R point mutation was correct,and the stable transformation cell line was verified by the real time-PCR or western blot. MTT results showed that the crizotinib drug resistant model was successfully constructed. Conclusion pEGFP-N1-EML4-ALK-G1202 R eukaryotic expression vector was successfully constructed,and EML4-ALK-G1202 R can be stably expressed after being transfected to A549 cell,and could develop the drug resistance of crizotinib. Our experiment laid a foundation for further mechanism study on EML4-ALK mutations-leaded cell resistance.
引文
[1]SIEGEL R,DESANTIS C,JEMAL A.Colorectal cancer statistics,2014[J].CA Cancer J Clin,2014,64(2):104-117.
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[7]CAMIDGE D R,BANG Y J,KWAK E L,et al.Activity and safety of crizotinib in patients w ith ALKpositive non-small cell lung cancer:updated results from a phase 1 study[J].Lancet Oncol,2012,13(10):1011-1119.
[8]DOEBELE R C,PILLING A B,AISNER D L,et al.M echanisms of resistance to crizotinib in patients w ith ALK gene rearranged non-small cell lung cancer[J].Clin Cancer Res,2012,18(5):1472-1482.
[9]KATAYAMA R,SHAW A T,KHAN T M,et al.M echanisms of acquired crizotinib resistance in ALKrearranged lung cancers[J].Sci Transl M ed,2012,4(120):120ra17.
[10]MIYAZAKI R,ANAYAMA T,HIROHASHI K,et al.In vitro drug sensitivity tests to predict molecular target drug responses in surgically resected lung cancer[J].PLo S One,2016,11(4):e0152665.
[11]TUMATI V,KUMAR S,YU L,et al.Effect of PF-02341066 and radiation on non-small cell lung cancer cells[J].Oncol Rep,2013,29(3):1094-1100.
[12]SODA M,CHOI Y L,ENOMOTO M,et al.Identification of the transforming EM L4-ALK fusion gene in non-small-cell lung cancer[J].Nature,2007,448(7153):561-566.
[13]SHAW A T,OU S H,BANG Y J,et al.Crizotinib in ROS1-rearranged non-small-cell lung cancer[J].The New England Journal of M edicine,2014,371(21):1963-1971.
[14]KATAYAMA R,KHAN TM,BENES C,et al.Therapeutic strategies to overcome crizotinib resistance in non-small cell lung cancers harboring the fusion oncogene EM L4-ALK[J].Proc Natl Acad Sci USA,2011,108(18):7535-7540.
[15]KRIS M G,JOHNSON B E,KWIATKOWSKI D J,et al.Identification of driver mutations in tumor specimens from 1,000 patients w ith lung adenocarcinoma:The NCI’s Lung Cancer M utation Consortium(LCM C)[J].J Clin Oncol,2011,29(suppl):7506.
[16]VOENA C,VARESIO L M,ZHANG L,et al.Oncogenic ALK regulates EM T in non-small cell lung carcinoma through repression of the epithelial splicing regulatory protein 1[J].Oncotarget,2016,7(22):33316-33330.
[2]DESANTIS C E,LIN C C,MAR IOTTO A B,et al.Cancer treatment and survivorship statistics,2014[J].CA Cancer J Clin,2014,64(4):252-271.
[3]DAVIDSON M R,GAZDAR A F,CLARKE B E.The pivotal role of pathology in the management of lung cancer[J].J Thorac Dis,2013,55(4):63-78.
[4]SHAW A T,KIM D W,NAKAGAWA K,et al.Crizotinib versus chemotherapy in advanced ALK-positive lung cancer[J].N Engl J M ed,2013,368(25):2385-2394.
[5]XU J,JIN B,CHU T,et al.EGFR tyrosine kinase inhibitor(TKI)in patients w ith advanced non-small cell lung cancer(NSCLC)harboring uncommon EGFR mutations:a real-w orld study in China[J].Lung Cancer,2016,96:87-92.
[6]WONG D W,LEUNG E L,SO K K,et al.The EM L4-ALK fusion gene is involved in various histologic types of lung cancers from nonsmokers w ith w ild-type EGFR and KRAS[J].Cancer,2009,115(8):1723-1733.
[7]CAMIDGE D R,BANG Y J,KWAK E L,et al.Activity and safety of crizotinib in patients w ith ALKpositive non-small cell lung cancer:updated results from a phase 1 study[J].Lancet Oncol,2012,13(10):1011-1119.
[8]DOEBELE R C,PILLING A B,AISNER D L,et al.M echanisms of resistance to crizotinib in patients w ith ALK gene rearranged non-small cell lung cancer[J].Clin Cancer Res,2012,18(5):1472-1482.
[9]KATAYAMA R,SHAW A T,KHAN T M,et al.M echanisms of acquired crizotinib resistance in ALKrearranged lung cancers[J].Sci Transl M ed,2012,4(120):120ra17.
[10]MIYAZAKI R,ANAYAMA T,HIROHASHI K,et al.In vitro drug sensitivity tests to predict molecular target drug responses in surgically resected lung cancer[J].PLo S One,2016,11(4):e0152665.
[11]TUMATI V,KUMAR S,YU L,et al.Effect of PF-02341066 and radiation on non-small cell lung cancer cells[J].Oncol Rep,2013,29(3):1094-1100.
[12]SODA M,CHOI Y L,ENOMOTO M,et al.Identification of the transforming EM L4-ALK fusion gene in non-small-cell lung cancer[J].Nature,2007,448(7153):561-566.
[13]SHAW A T,OU S H,BANG Y J,et al.Crizotinib in ROS1-rearranged non-small-cell lung cancer[J].The New England Journal of M edicine,2014,371(21):1963-1971.
[14]KATAYAMA R,KHAN TM,BENES C,et al.Therapeutic strategies to overcome crizotinib resistance in non-small cell lung cancers harboring the fusion oncogene EM L4-ALK[J].Proc Natl Acad Sci USA,2011,108(18):7535-7540.
[15]KRIS M G,JOHNSON B E,KWIATKOWSKI D J,et al.Identification of driver mutations in tumor specimens from 1,000 patients w ith lung adenocarcinoma:The NCI’s Lung Cancer M utation Consortium(LCM C)[J].J Clin Oncol,2011,29(suppl):7506.
[16]VOENA C,VARESIO L M,ZHANG L,et al.Oncogenic ALK regulates EM T in non-small cell lung carcinoma through repression of the epithelial splicing regulatory protein 1[J].Oncotarget,2016,7(22):33316-33330.