BMXΔN通过ERK/MAPK信号通路影响肺癌细胞对吉非替尼的耐药性

摘要/Abstract

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、2.00 μmol/L、4.00 μmol/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、1.00 μmol/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]、2.00 μmol/L[(70.22±3.45)%vs.(53.14±0.89)%,t=-8.309,P=0.001]、4.00 μmol/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]、1.00 μmol/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±0.06vs.0.38±0.12vs.0.37±0.14)、4 h(1.28±0.06vs.1.08±0.06vs.1.11±0.07)、8 h(0.75±0.04vs.0.55±0.05vs.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。

关键词:肺肿瘤,细胞增殖,BMXΔN,吉非替尼耐药,上皮间质转化

Abstract:

ObjectiveTo explore the mechanism of a novel BMX splicing variant induced epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) gefitinib resistance in lung cancer.MethodsStable 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, 4.00 μmol/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 1.00 μmol/L gefitinib. MTT method was used to detect cell viabilities.ResultsThe 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], 2.00 μmol/L [(70.22±3.45)%vs.(53.14±0.89)%,t=-8.309,P=0.001], 4.00 μmol/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], 1.00 μmol/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±0.04vs.0.81±0.04vs.0.80±0.05, allP<0.05); the expression levels of p-ERK1/2 in PC9-BMXΔN cells were significantly increased after gefitinib treatment for 2 h (0.64±0.06vs.0.38±0.12vs.0.37±0.14), 4 h (1.28±0.06vs.1.08±0.06vs.1.11±0.07), and 8 h (0.75±0.04vs.0.55±0.05vs.0.60±0.07), with statistically significant differences (allP<0.05).ConclusionBMXΔN 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.

Key words:Lung neoplasms,Cell proliferation,BMXΔN,Gefitinib resistance,Epithelial-mesenchymal transition

阎星羽, 廉振颖, 刁玉涛, 刘红艳. BMXΔN通过ERK/MAPK信号通路影响肺癌细胞对吉非替尼的耐药性[J]. 国际肿瘤学杂志, 2021, 48(6): 328-334.

Yan Xingyu, Lian Zhenying, Diao Yutao, Liu Hongyan. BMXΔN mediates gefitinib resistance of lung cancer cells through ERK/MAPK signaling pathway[J]. Journal of International Oncology, 2021, 48(6): 328-334.

图/表8

表1

引物序列"

基因名称	引物序列
GAPDH	正向引物:5'-GCGACACCCACTCCTCCACCTTT-3'
	反向引物:5'-TGCTGTAGCCAAATTCGTTGTCATA-3'
纤连蛋白	正向引物:5'-GGACCTACCTAGGCAATGCG-3'
	反向引物:5'-ATGGCAGCGGTTTGCGAT-3'
神经钙黏素	正向引物:5'-ATGCTGACGATCCCAATGC-3'
	反向引物:5'-GCCTTCCATGTCTGTAGCTTGA-3'
上皮钙黏素	正向引物:5'-CCTGGACGCTCGGCCTGAAG-3'
	反向引物:5'-ATAAGGCGGGGCTGTGGGGT-3'
波形蛋白	正向引物:5'-CCTTCGTGAATACCAAGACCTG-3'
	反向引物:5'-CTGCTCTCCTCGCCTTCC-3'
Snail	正向引物:5'-CTGCCCTGCGTCTGCGGAAC-3'
	反向引物:5'-GGAGCGGTCAGCGAAGGCAC-3'
Slug	正向引物:5'-AGACCCCCATGCCATTGAAG-3'
	反向引物:5'-GGCCAGCCCAGAAAAAGTTG-3'
TWIST 2	正向引物:5'-CCCACCTCTGCCCTCCACCA-3'
	反向引物:5'-GCAAGAGGGCTGGGAGTGCC-3'

表1

图1

组别	纤连蛋白	神经钙黏素	上皮钙黏素	波形蛋白	Snail	Slug	TWIST 2
PC9-Vec组	0.12±0.02	0.04±0.00	0.47±0.03	0.03±0.01	0.03±0.01	0.10±0.01	0.07±0.02
PC9-BMXΔN组	0.30±0.01	0.13±0.01	0.47±0.03	0.06±0.01	0.13±0.06	0.17±0.04	0.15±0.02
t值	-15.494	-22.933	0.272	-5.117	-3.985	-2.909	-4.697
P值	<0.001	<0.001	0.799	0.007	0.016	0.044	0.009

表2

组别	纤连蛋白	上皮钙黏素	波形蛋白
PC9-Vec组	0.96±0.03	1.07±0.05	0.75±0.04
PC9-BMX组	1.05±0.05^a	1.02±0.04^b	0.74±0.03^c
PC9-BMXΔN组	1.20±0.07^d	0.91±0.03^e	1.11±0.05^f
F值	15.765	11.174	72.836
P值	0.004	0.009	<0.001

表3

图2

组别	吉非替尼浓度
PC9-Vec组	100.00±5.49	91.29±1.91	63.36±2.14	60.22±3.61	59.93±1.91	56.70±2.88	53.14±0.89	52.30±2.59
PC9-BMXΔN组	100.00±3.39	99.11±2.16	80.41±1.48	80.83±5.38	75.54±3.46	77.57±6.53	70.22±3.45	68.66±4.67
t值	<0.001	-4.701	-11.324	-5.507	-6.836	-5.064	-8.309	-4.882
P值	>0.999	0.011	<0.001	0.005	0.002	0.007	0.001	0.008

表4

组别	吉非替尼浓度
HCC827-Vec组	100.00±4.51	78.17±2.18	47.13±4.21	43.28±2.95	37.12±4.92	32.06±4.73
HCC827-BMXΔN组	100.00±8.30	78.80±8.67	64.36±2.49	63.25±5.87	49.47±5.74	49.05±3.34
t值	<0.001	-0.121	-7.067	-5.267	-2.830	-5.073
P值	>0.999	0.908	0.019	0.006	0.047	0.007

表5

表6

吉非替尼处理的各组人肺癌细胞株PC9细胞中p-EGFR和p-ERK1/2蛋白相对表达水平比较(x?±s,n=3)"

组别	p-EGFR							F值	P值	p-ERK1/2								F值		P值
组别	0 h		2 h		4 h		8 h	F值	P值	0 h		2 h		4 h		8 h		F值		P值
PC9-Vec组	1.80±0.07	0.96±0.06^a		0.80±0.06^a		0.81±0.04^a		221.462	<0.001	1.93±0.07	0.38±0.12^a		1.08±0.06^a		0.55±0.05^a		219.659		<0.001
PC9-BMX组	1.78±0.08	0.96±0.06^a		0.80±0.05^a		0.80±0.05^ab		185.942	<0.001	1.93±0.05	0.37±0.14^ab		1.11±0.07^ab		0.60±0.07^ab		172.368		<0.001
PC9-BMXΔN组	1.81±0.08	0.97±0.04^a		0.83±0.05^a		0.91±0.04^ac		211.687	<0.001	1.94±0.05	0.64±0.06^ac		1.28±0.06^ac		0.75±0.04^ac		377.478		<0.001
F值	0.073	0.045		0.334		7.156				0.018	5.640		8.302		11.600
P值	0.930	0.956		0.729		0.026				0.983	0.042		0.019		0.009

表6

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