Abstract
We previously reported that the expression of G protein‐coupled receptor kinase 6 (GRK6) is significantly downregulated in lung adenocarcinoma (LADC) tissues, and low expression levels of GRK6 are correlated with poor survival prognosis. However, the specific regulatory mechanisms and functions of GRK6 in LADC remain unknown. Here, we report that GRK6 mRNA expression levels are downregulated in LADC tissues compared to those in matched adjacent non‐tumor tissues (P < 0.001). The promoter of the GRK6 gene was found to be hypermethylated in LADC tissues, and its methylation was correlated with both GRK6 expression and pathology grade. GRK6 promoter hypermethylation may predict shorter overall survival. Treatment with 5‐aza‐2′‐deoxycytidine significantly enhanced GRK6 gene expression. Four binding sites of CCAAT/enhancer‐binding protein‐α (C/EBPα) in the CpG island of the GRK6 gene promoter were predicted in silico, of which three sites were further confirmed by ChIP. Decreased binding of C/EBPα to binding sites 1, 3 and 4 of the GRK6 gene promoter was observed in LADC tissues. Inhibition of C/EBPα significantly inhibited GRK6 expression, while overexpression of C/EBPα significantly promoted GRK6 expression. In addition, overexpression of GRK6 significantly suppressed, while GRK6 knockdown promoted cell migration and invasion. Overexpression of GRK6 enhanced E‐cadherin expression and suppressed vimentin expression, and silencing of GRK6 had the opposite effects. Furthermore, ectopic expression of GRK6 significantly decreased matrix metalloproteinase (MMP) 2 and MMP7 protein expression levels. Our findings suggest that hypermethylation of the GRK6 gene promoter suppressed binding of C/EBPα, thereby contributing to the promotion of cell migration and invasion. The methylation status of the GRK6 promoter might be suitable for use as an epigenetic biomarker, and the C/EBPα–GRK6 signaling pathway may be a potential target for LADC.
Highlights
We previously reported that the expression of G protein-coupled receptor kinase 6 (GRK6) is significantly downregulated in lung adenocarcinoma (LADC) tissues, and low expression levels of GRK6 are correlated with poor survival prognosis
We found a decrease trend in GRK6 expression levels in LADC tissues compared with those in non-tumor tissues using The Cancer Genome Atlas (TCGA) database analysis (Fig. 1B)
The results indicated that CCAAT/enhancerbinding protein-a (C/EBPa) can bind to the CpG island of the GRK6 gene promoter at S1, S3 and S4, whereas no binding was observed at S2 (Fig. 5B)
Summary
We previously reported that the expression of G protein-coupled receptor kinase 6 (GRK6) is significantly downregulated in lung adenocarcinoma (LADC) tissues, and low expression levels of GRK6 are correlated with poor survival prognosis. The promoter of the GRK6 gene was found to be hypermethylated in LADC tissues, and its methylation was correlated with both GRK6 expression and pathology grade. Abbreviations 5-Aza-CdR, 5-aza-20-deoxycytidine; BSP, bisulfite sequencing PCR; C/EBPa, CCAAT/enhancer-binding protein-a; EMT, epithelial– mesenchymal transition; GRK6, G protein-coupled receptor kinase 6; GRK, G protein-coupled receptor kinase; LADC, lung adenocarcinoma; MMP, matrix metalloproteinase; MSP, methylation-specific PCR; NSCLC, non-small-cell lung cancer; qPCR, quantitative PCR. G protein-coupled receptor kinase 6 (GRK6), a GRK family member, has previously been shown to play a critical role in many diseases process [12,13,14]. The specific regulatory mechanisms and functions of GRK6 in LADC remain to be examined
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