PAR2 Promoter Hypomethylation Regulates PAR2 Gene Expression and Promotes Lung Adenocarcinoma Cell Progression

Abstract

Objective: Protease-activated receptor-2 (PAR2) also known as F2RL1 is a G protein-coupled receptor that intimately correlates with cancer occurrence. DNA methylation turns out a vital mechanism regulating gene expression, while PAR2 promoter methylation is proven to be involved in cancer development. Hence, this study attempted to clarify the molecular mechanism by which PAR2 mediates lung adenocarcinoma (LUAD) progression, via identifying the effect of PAR2 promoter methylation on LUAD cell progression.

Methods: Associations of PAR2 promoter methylation with PAR2 gene expression and prognosis of LUAD patients were analyzed via bioinformatics analysis. PAR2 promoter methylation and gene expression at the cellular level were measured using methylation-specific PCR, qRT-PCR, and Western blot assays. DNA methyltransferase inhibitor 5-AzadC was used to treat cells to assess PAR2 gene expression alteration. Cell biological behaviors upon PAR2 overexpression were characterized via MTT, wound healing assay, and Transwell assay.

Results: Bioinformatics analysis revealed that PAR2 promoter methylation was negatively related to PAR2 gene expression, while PAR2 promoter hypermethylation and low gene expression indicated favorable LUAD prognosis. Besides, it turned out that PAR2 presented upregulated expression and hypomethylated promoter in LUAD cells. Moreover, PAR2 gene expression was elevated in cells treated with 5-AzadC, and the proliferative, migratory, and invasive capabilities of cells with 5-AzadC or high PAR2 gene expression were all enhanced.

Conclusion: In sum, PAR2 promoter hypomethylation potentiates LUAD cell progression, in turn affecting the prognosis of LUAD patients.

Figure 1

Bioinformatics analysis suggests that PAR2 promoter methylation is related to PAR2 mRNA expression and the prognosis of LUAD patients. (a) “MethylMix” package was used to screen methylation-driven genes from TCGA-LUAD dataset as plotted in a heat map, with red representing high methylation and green representing low methylation. PAR2 was identified and its (b) promoter methylation level was detected in tumor samples (histogram) and normal tissue samples (horizontal black line) in TCGA-LUAD dataset. (c) Correlation analysis for PAR2 promoter methylation and gene expression. (d) Relative expression level of PAR2 in TCGA was examined. (e) Survival analysis shows overall survival difference between high/low PAR2 promoter methylation level in LUAD patients (red line: hypermethylation; blue line: hypomethylation). (f) Overall survival difference in LUAD patients between hyper- and low-expression (red line) and hypo- and high-expression (blue line) groups. Afterwards, (g) the promoter methylation level of PAR2 was determined in different tumor stages and T stages combined with clinical information. (h) Correlation analysis between methylation level of three PAR2-related methylated sites and PAR2 expression level, respectively.

Figure 2

PAR2 has increased mRNA expression and decreased promoter methylation in LUAD cells. (a) qRT-PCR and (b) Western blot results show the mRNA and protein expression of PAR2 at the cellular level. (c) MSP was carried out to determine the methylation of PAR2 promoter in each cell line (U: unmethylated; M: methylated). (^∗p < 0.05).

Figure 3

PAR2 promoter hypomethylation promotes PAR2 expression in LUAD cells. (a) qRT-PCR and (b) Western blot results present PAR2 mRNA and protein levels in 5-AzadC-treated cells. (^∗∗p < 0.01).

Figure 4

PAR2 promoter hypomethylation or PAR2 mRNA overexpression potentiates LUAD cell malignant progression. 5-AzadC, oe-PAR2, and their controls were used to treat H1299 and A549 cells. Cells were harvested for (a) MTT, (b) wound healing assay, and (c, 100x) Transwell assay to assess cell proliferative, migratory, and invasive abilities. (^∗p < 0.05, ^∗∗p < 0.01).