GPR87 promotes invasion and migration through the RHO/ROCK pathway in non-small cell lung cancer

doi:10.3969/j.issn.1674-8115.2024.12.004

Abstract

Abstract:

Objective ·To explore the role and molecular mechanism of GPR87 in regulating the invasion and migration of non-small cell lung cancer (NSCLC). Methods ·Bioinformatics methods, including GEO, UALCAN, KM Plotter and other public database analysis platforms, were used to screen candidate genes related to NSCLC invasion and predict their clinical relevance to NSCLC. Eighty NSCLC clinical patient samples and corresponding clinical pathological data were collected from Yichang Central People's Hospital from January 2018 to August 2020. Immunohistochemistry was used to analyze the expression of GPR87 in tumor tissues and the clinical relevance of GPR87 was analyzed. siRNA-GPR87 and pCMV-GPR87-his were transfected into the human lung adenocarcinoma cell line A549 and the human lung squamous cell carcinoma cell line SK-MES-1, to construct cell lines with low and high expression of GPR87. Transwell assay was used to investigate the effect of GPR87 expression on the migration and invasion ability of NSCLC cells. ELISA was used to detect the secretion of MMP7 in the culture supernatant. RT-qPCR was used to detect the mRNA expression levels of GPR87, MMP2, MMP7, MMP9, E-cadherin,N-cadherin, vimentin,snail,twist, RHOA, RHOC, and ROCK1. ELISA was used to detect the secreted protein MMP7. Western blotting was used to detect the protein expression levels ofGPR87, MMP9, E-cadherin, vimentin, RHOA, and ROCK1. Results ·Bioinformatics analysis of clinical sample data showed that GPR87 was highly expressed in NSCLC. Patients with higher expression of GPR87 had worse clinical stage and were more prone to lymph node metastasis, suggesting that GPR87 might be a key gene for the high invasiveness of NSCLC. Downregulation of GPR87 expression significantly reduced the invasion and migration ability of A549 and SK-MES-1 cells, while overexpression of GPR87 enhanced the invasion and migration ability of A549 and SK-MES-1 cells. Further detection revealed that downregulation of GPR87 led to decreased mRNA expression levels of MMP2, MMP7, MMP9, RHOA, RHOC, and ROCK1, as well as a reduction in the secretion of MMP7 and the protein expression levels of MMP9, RHOA, and ROCK1 in A549 and SK-MES-1 cells. Overexpression of GPR87 increased the mRNA expression levels of MMP2, MMP7, MMP9, RHOA, RHOC, and ROCK1, as well as the secretion of MMP7 and the protein expression levels of MMP9, RHOA, and ROCK1. Regardless of GPR87 knockdown or overexpression, the expression of genes and proteins related to epithelial-mesenchymal transition (EMT) in the cells did not change significantly. Conclusion ·High expression of GPR87 is closely related to the high invasiveness of NSCLC. In SK-MES-1 and A549 cells, GPR87 can activate the RHOA/ROCK1 signaling pathway, promote the expression of MMPs, and ultimately promote the invasion and migration of NSCLC.

Key words: GPR87, non-small cell lung cancer (NSCLC), invasion, migration, RHO/ROCK signaling pathway

CLC Number:

LIU Chenxi, HAN Lin, YANG Yi, ZHOU Han, LIU Yayun, SHENG Deqiao. GPR87 promotes invasion and migration through the RHO/ROCK pathway in non-small cell lung cancer[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2024, 44(12): 1514-1525.

Figures/Tables 12

TrendMD

References 21

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Name	Forward (5'→3')	Reverse (5'→3')
siRNA-GPR87-1	GACCUUAGUUUCAAAGCUUdTdT	AAGCUUUGAAACUAAGGUCdTdT
siRNA-GPR87-2	GCAUCUUGCUGAAUGGUUUdTdT	AAACCAUUCAGCAAGAUGCdTdT
NC-siRNA	UUCUCCGAACGUGUCACGUTT	ACGUGACACGUUCGGAGAATT

Name	Forward (5'→3')	Reverse (5'→3')
siRNA-GPR87-1	GACCUUAGUUUCAAAGCUUdTdT	AAGCUUUGAAACUAAGGUCdTdT
siRNA-GPR87-2	GCAUCUUGCUGAAUGGUUUdTdT	AAACCAUUCAGCAAGAUGCdTdT
NC-siRNA	UUCUCCGAACGUGUCACGUTT	ACGUGACACGUUCGGAGAATT

Gene	Forward (5'→3')	Reverse (5'→3')
GPR87	GGAGGCGACATCAATGCAG	AAATGAAAGTAAAGAACGATTTTGTGT
MMP2	GCTGGAGACAAATTCTGGAGATACA	GTATCGAAGGCAGTGGAGAGGA
MMP7	GAGTGAGCTACAGTGGGAACA	CTATGACGCGGGAGTTTAACAT
MMP9	GCCACTACTGTGCCTTTGAGTC	CCCTCAGAGAATCGCCAGTACT
E-cadherin	GCCTCCTGAAAAGAGAGTGGAAG	TGGCAGTGTCTCTCCAAATCCG
N-cadherin	CCTCCAGAGTTTACTGCCATGAC	GTAGGATCTCCGCCACTGATTC
vimentin	AGGCAAAGCAGGAGTCCACTGA	ATCTGGCGTTCCAGGGACTCAT
snail	TGCCCTCAAGATGCACATCCGA	GGGACAGGAGAAGGGCTTCTC
twist	GCCAGGTACATCGACTTCCTCT	TCCATCCTCCAGACCGAGAAGG
RHOA	GCAGGTAGAGTTGGCTTTATGG	CTTGTGTGCTCATCATTCCGA
RHOC	AAGACGAGCACACCAGGAGAGA	TTGGCTGAGCACTCAAGGTAGC
ROCK1	GAAACAGTGTTCCATGCTAGACG	GCCGCTTATTTGATTCCTGCTCC
β-actin	CTGGAACGGTGAAGGTGACA	AAGGGACTTCCTGTAACAACGCA

Gene	Forward (5'→3')	Reverse (5'→3')
GPR87	GGAGGCGACATCAATGCAG	AAATGAAAGTAAAGAACGATTTTGTGT
MMP2	GCTGGAGACAAATTCTGGAGATACA	GTATCGAAGGCAGTGGAGAGGA
MMP7	GAGTGAGCTACAGTGGGAACA	CTATGACGCGGGAGTTTAACAT
MMP9	GCCACTACTGTGCCTTTGAGTC	CCCTCAGAGAATCGCCAGTACT
E-cadherin	GCCTCCTGAAAAGAGAGTGGAAG	TGGCAGTGTCTCTCCAAATCCG
N-cadherin	CCTCCAGAGTTTACTGCCATGAC	GTAGGATCTCCGCCACTGATTC
vimentin	AGGCAAAGCAGGAGTCCACTGA	ATCTGGCGTTCCAGGGACTCAT
snail	TGCCCTCAAGATGCACATCCGA	GGGACAGGAGAAGGGCTTCTC
twist	GCCAGGTACATCGACTTCCTCT	TCCATCCTCCAGACCGAGAAGG
RHOA	GCAGGTAGAGTTGGCTTTATGG	CTTGTGTGCTCATCATTCCGA
RHOC	AAGACGAGCACACCAGGAGAGA	TTGGCTGAGCACTCAAGGTAGC
ROCK1	GAAACAGTGTTCCATGCTAGACG	GCCGCTTATTTGATTCCTGCTCC
β-actin	CTGGAACGGTGAAGGTGACA	AAGGGACTTCCTGTAACAACGCA

ID	P_adj	P value	logFC	Gene symbol
219936_s_at	3.47×10^-5	2.32×10^-7	2.43	GPR87
212094_at	1.24×10^-5	4.70×10^-8	2.36	PEG10
209278_s_at	1.63×10^-2	1.53×10^-3	2.22	TFPI2
227506_at	1.25×10^-5	5.17×10^-8	2.14	SLC16A9
206023_at	3.69×10^-4	6.62×10^-6	2.12	NMU
205048_s_at	3.22×10^-3	1.44×10^-4	2.02	PSPH
220393_at	7.91×10^-3	5.37×10^-4	-2.05	LGSN
213432_at	1.33×10^-3	3.96×10^-5	-2.17	MUC5B
225728_at	2.93×10^-4	4.65×10^-6	-2.23	SORBS2
203824_at	1.38×10^-3	4.19×10^-5	-2.25	TSPAN8