摘要
目的:探讨一种BMX可变剪切体BMXΔN参与肺癌表皮生长因子受体酪氨酸激酶抑制剂(EGFR-TKI)吉非替尼耐药的作用机制。方法:利用慢病毒感染携带EGFR突变的人肺癌细胞株PC9和HCC827构建BMXΔN稳转细胞株。实验分为PC9-Vec组(阴性对照转染空载体PC9细胞)、PC9-BMX组(稳定表达BMX的PC9细胞)、PC9-BMXΔN组(稳定表达BMXΔN的PC9细胞)以及HCC827-Vec组(阴性对照转染空载体HCC827细胞)、HCC827-BMXΔN组(稳定表达BMXΔN的HCC827细胞)。采用实时定量PCR检测细胞中mRNA表达水平;采用蛋白质印迹法检测细胞中蛋白表达水平;0 nmol/L、0.01 nmol/L、2.00 nmol/L、50.00 nmol/L、100.00 nmol/L、200.00 nmol/L、2.00μmol/L、4.00μmol/L吉非替尼处理PC9-Vec组和PC9-BMXΔN组细胞,0 nmol/L、0.01 nmol/L、1.00 nmol/L、10.00 nmol/L、100.00 nmol/L、1.00μmol/L吉非替尼处理HCC827-Vec组和HCC827-BMXΔN组细胞,采用MTT法检测细胞活力。结果:PC9-BMXΔN组细胞散落并呈现成纤维样形态。与PC9-Vec组细胞相比,PC9-BMXΔN组细胞中纤连蛋白、神经钙黏素、波形蛋白、Snail、Slug和TWIST 2的mRNA表达均上调。与PC9-Vec组和PC9-BMX组细胞相比,PC9-BMXΔN组细胞中纤连蛋白和波形蛋白表达均上调,上皮钙黏素的表达下调。与PC9-Vec组细胞相比,经0.01 nmol/L[(99.11±2.16)%vs.(91.29±1.91)%,t=-4.701,P=0.011]、2.00 nmol/L[(80.41±1.48)%vs.(63.36±2.14)%,t=-11.324,P<0.001]、50.00 nmol/L[(80.83±5.38)%vs.(60.22±3.61)%,t=-5.507,P=0.005]、100.00 nmol/L[(75.54±3.46)%vs.(59.93±1.91)%,t=-6.836,P=0.002]、200.00 nmol/L[(77.57±6.53)%vs.(56.70±2.88)%,t=-5.064,P=0.007]、2.00μmol/L[(70.22±3.45)%vs.(53.14±0.89)%,t=-8.309,P=0.001]、4.00μmol/L[(68.66±4.67)%vs.(52.30±2.59)%,t=-4.882,P=0.008]浓度吉非替尼处理的PC9-BMXΔN组细胞活力均显著升高,差异均有统计学意义;与HCC827-Vec组相比,经1.00 nmol/L[(64.36±2.49)%vs.(47.13±4.21)%,t=-7.067,P=0.019]、10.00 nmol/L[(63.25±5.87)%vs.(43.28±2.95)%,t=-5.267,P=0.006]、100.00 nmol/L[(49.47±5.74)%vs.(37.12±4.92)%,t=-2.830,P=0.047]、1.00μmol/L[(49.05±3.34)%vs.(32.06±4.73)%,t=-5.073,P=0.007]吉非替尼处理的HCC827-BMXΔN组细胞活力均显著升高,差异均具有统计学意义。吉非替尼处理明显抑制了PC9-Vec组、PC9-BXM组和PC9-BMXΔN组细胞中p-EGFR和p-ERK1/2的表达;与PC9-Vec组(0.81±0.04)和PC9-BXM组(0.80±0.05)相比,PC9-BMXΔN组细胞p-EGFR的表达水平经吉非替尼处理8 h后(0.91±0.04)显著升高(均P<0.05);p-ERK1/2的表达经吉非替尼处理2 h后(0.64±0.06 vs.0.38±0.12 vs.0.37±0.14)、4 h(1.28±0.06 vs.1.08±0.06 vs.1.11±0.07)、8 h(0.75±0.04 vs.0.55±0.05 vs.0.60±0.07)均显著升高,差异均具有统计学意义(均P<0.05)。结论:BMXΔN参与了肺癌EGFR-TKI吉非替尼耐药,可能是通过诱导细胞发生上皮间质转化和激活ERK/MAPK通路实现的。
Objective To explore the mechanism of a novel BMX splicing variant induced epidermal growth factor receptor tyrosine kinase inhibitor(EGFR-TKI)gefitinib resistance in lung cancer.Methods Stable transgenic cell line PC9-BMXΔN and HCC827-BMXΔN were constructed by lentivirus infection of PC9 and HCC827 cells carrying EGFR mutation.The cells were divided into PC9-Vec group(PC9 cells transfected with empty vector),PC9-BMX group(PC9 cells stably expressing BMX),PC9-BMXΔN group(PC9 cells stably expressing BMXΔN)and HCC827-Vec group(HCC827 cells transfected with empty vector),HCC827-BMXΔN group(HCC827 cells stably expressing BMXΔN).Quantitative real-time PCR was used to detect the expression levels of mRNA.The protein expression levels in each group were detected by Western blotting.The cells in the PC9-Vec group and PC9-BMXΔN group were treated with 0,0.01,2.00,50.00,100.00,200.00 nmol/L and 2.00,4.00μmol/L gefitinib.The cells in the HCC827-Vec group and HCC827-BMXΔN group were treated with 0,0.01,1.00,10.00,100.00 nmol/L and 1.00μmol/L gefitinib.MTT method was used to detect cell viabilities.Results The PC9-BMXΔN cells were scattered and showed a fibroblast-like morphology.Compared with the PC9-Vec cells,the relative expression levels of fibronectin,N-cadherin,vimentin,Snail,Slug and TWIST 2 mRNA in PC9-BMXΔN cells were up-regulated.Compared with the PC9-Vec cells and PC9-BMX cells,the expression levels of fibronectin and vimentin protein in PC9-BMXΔN cells were up-regulated;while the expression level of E-cadherin protein in PC9-BMXΔN cells was significantly down-regulated.Compared with the PC9-Vec cells,the cell viabilities of PC9-BMXΔN cells treated with 0.01 nmol/L[(99.11±2.16)%vs.(91.29±1.91)%,t=-4.701,P=0.011],2.00 nmol/L[(80.41±1.48)%vs.(63.36±2.14)%,t=-11.324,P<0.001],50.00 nmol/L[(80.83±5.38)%vs.(60.22±3.61)%,t=-5.507,P=0.005],100.00 nmol/L[(75.54±3.46)%vs.(59.93±1.91)%,t=-6.836,P=0.002],200.00 nmol/L[(77.57±6.53)%vs.(56.70±2.88)%,t=-5.064,P=0.007],2.00μmol/L[(70.22±3.45)%vs.(53.14±0.89)%,t=-8.309,P=0.001],4.00μmol/L[(68.66±4.67)%vs.(52.30±2.59)%,t=-4.882,P=0.008]gefitinib were significantly increased,with statistically significant differences.Similarly,compared with the HCC827-Vec cells,the cell viabilities of HCC827-BMXΔN cells treated with 1.00 nmol/L[(64.36±2.49)%vs.(47.13±4.21)%,t=-7.067,P=0.019],10.00 nmol/L[(63.25±5.87)%vs.(43.28±2.95)%,t=-5.267,P=0.006],100.00 nmol/L[(49.47±5.74)%vs.(37.12±4.92)%,t=-2.830,P=0.047],1.00μmol/L[(49.05±3.34)%vs.(32.06±4.73)%,t=-5.073,P=0.007]gefitinib were significantly increased,with statistically significant differences.Gefitinib treatment could significantly inhibit the expression levels of p-EGFR and p-ERK1/2 both in PC9-Vec cells,PC9-BMX cells and PC9-BMXΔN cells.Compared with the PC9-Vec cells and PC9-BMX cells,the expression level of p-EGFR in PC9-BMXΔN cells was significantly increased after gefitinib treatment for 8 h(0.91±0.04 vs.0.81±0.04 vs.0.80±0.05,all P<0.05);the expression levels of p-ERK1/2 in PC9-BMXΔN cells were significantly increased after gefitinib treatment for 2 h(0.64±0.06 vs.0.38±0.12 vs.0.37±0.14),4 h(1.28±0.06 vs.1.08±0.06 vs.1.11±0.07),and 8 h(0.75±0.04 vs.0.55±0.05 vs.0.60±0.07),with statistically significant differences(all P<0.05).Conclusion BMXΔN is involved in EGFR-TKI gefitinib resistance in lung cancer,which may be achieved by inducing cells to undergo epithelial-mesenchymal transition and activating the ERK/MAPK signaling pathway.
作者
阎星羽
廉振颖
刁玉涛
刘红艳
Yan Xingyu;Lian Zhenying;Diao Yutao;Liu Hongyan(School of Basic Medicine,Shandong First Medical University Institute of Basic Medicine,Shandong Academy of Medical Sciences,Jinan 250062,China;Research Center of Basic Medicine,Jinan Central Hospital,Shandong First Medical University,Jinan 250013,China)
出处
《国际肿瘤学杂志》
CAS
2021年第6期328-334,共7页
Journal of International Oncology
基金
山东省重点研发计划(2019GSF108185)。
关键词
肺肿瘤
细胞增殖
BMXΔN
吉非替尼耐药
上皮间质转化
Lung neoplasms
Cell proliferation
BMXΔN
Gefitinib resistance
Epithelial-mesenchymal transition
