BMXΔN mediates gefitinib resistance of lung cancer cells through ERK/MAPK signaling pathway

doi:10.3760/cma.j.cn371439-20201230-00063

Abstract

Abstract:

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, 4.00 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. 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], 2.00 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±0.04 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.

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

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.

Figures/Tables 8

References 14

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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'

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

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

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

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