Effect of autophagy induced by PDZ binding kinase on chemosensitivity of lung cancer cells to cisplatin

doi:10.3877/cma.j.issn.2095-1221.2021.04.005

Abstract

Abstract:

Objective

To investigate the effect of PDZ binding kinase (PBK) on the chemosensitivity of lung cancer cells to cisplatin and its related mechanism.

Methods

A549 cells were treated with cisplatin or transfected with si-NC, si-PBK, si-EVI1, oe-NC and oe-EVI1. The mRNA expressions of ecotropic viral integration site-1 (EVI1) and PBK in human lung cancer cell lines A549, HCC827, NCI-H1299 and BEAS-2B were detected by RT-qPCR; The protein expressions of EVI1, PBK, Beclin1, p62 and microtubule associated protein 1 light chain 3 (LC3B) were detected by Western blot; Cell viability was detected by CCK-8; cell proliferation was detected by EdU assay; Migration and invasion of cells were detected by Transwell assay; The binding of EVI1 and PBK promoter region was detected by ChIP-PCR. One-way analysis of variance was used to compare the difference among multiple groups, and LSD-t test was used to comparethe difference in the expression of EVI1 and PBK between HCC827 group and BEAS-2B group, NCI-H1299 group and BEAS-2B group, A549 and BEAS-2B group. Independent sample t-test was used to compare the difference in the expression of PBK between si-NC group and si-EVI1 group, oe-NC group and oe-EVI1 group.

Results

Compared with BEAS-2B cells, the expression of EVI1 and PBK mRNA and protein in HCC827, NCI-H1299 and A549 cells increased, and the difference was statistically significant (all P < 0.05) . Compared with the blank control group, the expression of EVI1 and PBK mRNA and protein were incread in the cells of the cisplatin group and the cisplatin+si-NC group, and the protein expression of Beclin1 and LC3Ⅱ/LC3Ⅰ (0.15±0.01 vs 0.36±0.02, 0.34±0.02) (1.32±0.11 vs 4.16±0.21, 4.04±0.15) was increased, the difference was statistically significant (P < 0.05) ; Howerer cell viability decreased at 24, 48, and 72 h, the rate of EdU positive cells [ (42.71±2.56) ﹪ vs (30.25±1.91) ﹪, (28.90±2.51) ﹪], migration number (133.67±6.51 vs 72.00±4.00, 70.00±2.65) , the number of invasions (81.00±4.00 vs 43.00±3.00, 42.00±3.00) decreased, the expression of p62 protein (0.65±0.03 vs 0.22±0.02, 0.23±0.01) were decreased, and the differences were statistically significant (all P < 0.05) . Compared with the cisplatin group and the cisplatin+si-NC group, the expression of PBK mRNA and protein was decreased in the cisplatin+si-PBK group decreased, and the cell viability was also decreased at 24, 48, and 72 h. The rate of EdU positive cells was [ (30.25±1.91) ﹪ vs (28.90±2.51) ﹪, (22.25±1.97) ﹪], migration number (72.00±4.00, 70.00±2.65 vs 34.00±3.00) , invasion number (43.00±3.00, 42.00±3.00 vs 21.33±2.52) , the expression of Beclin1 and LC3Ⅱ/LC3Ⅰ protein (0.36±0.02, 0.34±0.02 vs 0.25±0.02) (4.16±0.21, 4.04±0.15 vs 2.45±0.22) were decreased, and the difference were statistically significant (all P < 0.05) ; The expression of p62 protein (0.22±0.02, 0.23±0.01 vs 0.46±0.02) was increased, and the difference was statistically significant (P < 0.05) . Compared with the si-NC group, EVI1 directly binds to the PBK promoter region. Compared with the si-NC group, the expression of EVI1 and PBK mRNA and protein was decreased in the cells of the si-EVI1 group, and the difference was statistically significant (all P < 0.05) ; Compared with the oe-NC group, the expression of EVI1 and PBK mRNA and protein was increased in the cells of the oe-EVI1 group, and the difference was statistically significant (all P <0.05) .

Conclusion

PBK regulated by EVI1 may inhibit the chemotherapy sensitivity of lung cancer cells to cisplatin by promoting autophagy.

Key words: Lung cancer, PDZ-binding kinase, Ecotropic viral integration site-1, Autophagy, Cisplatin

Xiaoli Yuan, Yang Li, Wei Deng, Shijuan Nie. Effect of autophagy induced by PDZ binding kinase on chemosensitivity of lung cancer cells to cisplatin[J]. Chinese Journal of Cell and Stem Cell(Electronic Edition), 2021, 11(04): 222-230.

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Figures/Tables 15

References 23

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基因	引物序列（5'→3'）	预期扩增产物长度（bp）
EVI1	上游CTCAGCATTTTCAATGGTTGA	147
	下游TGTGACAGCATGTGTTTCTCC
PBK	上游TATGGCAAGAGGGTTAAAGTATCTGC	176
	下游CAATGTAACAAGCCTCAGGGTCAG
GAPDH	上游GGTCACCAGGGCTGCTTTTA	141
	下游GGATCTCGCTCCTGGAAGATG

基因	引物序列（5'→3'）	预期扩增产物长度（bp）
EVI1	上游CTCAGCATTTTCAATGGTTGA	147
	下游TGTGACAGCATGTGTTTCTCC
PBK	上游TATGGCAAGAGGGTTAAAGTATCTGC	176
	下游CAATGTAACAAGCCTCAGGGTCAG
GAPDH	上游GGTCACCAGGGCTGCTTTTA	141
	下游GGATCTCGCTCCTGGAAGATG

分组	EVI1 mRNA表达	EVI1蛋白表达	PBK mRNA表达	PBK蛋白表达
BEAS-2B	1.00±0.05	0.18±0.01	1.00±0.06	0.15±0.01
HCC827	2.23±0.22^a	0.32±0.01^a	2.54±0.20^a	0.29±0.01^a
NCI-H1299	2.65±0.15^a	0.35±0.02^a	2.75±0.13^a	0.36±0.02^a
A549	2.88±0.15^a	0.51±0.02^a	3.22±0.19^a	0.57±0.03^a
F值	88.129	209.967	113.865	248.899
P值	< 0.001	< 0.001	< 0.001	< 0.001

分组	EVI1 mRNA表达	EVI1蛋白表达	PBK mRNA表达	PBK蛋白表达
BEAS-2B	1.00±0.05	0.18±0.01	1.00±0.06	0.15±0.01
HCC827	2.23±0.22^a	0.32±0.01^a	2.54±0.20^a	0.29±0.01^a
NCI-H1299	2.65±0.15^a	0.35±0.02^a	2.75±0.13^a	0.36±0.02^a
A549	2.88±0.15^a	0.51±0.02^a	3.22±0.19^a	0.57±0.03^a
F值	88.129	209.967	113.865	248.899
P值	< 0.001	< 0.001	< 0.001	< 0.001

分组	PBK mRNA表达	PBK蛋白表达
空白对照	1.00±0.06	0.46±0.02
顺铂	2.24±0.11^a	0.89±0.02^a
顺铂+si-NC	2.29±0.10^a	0.88±0.03^a
顺铂+si- PBK	0.34±0.02^abc	0.18±0.01^abc
F值	399.108	496.115
P值	< 0.001	< 0.001

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