Journal: FEBS Open Bio

Article Title: Hypermethylation of the G protein‐coupled receptor kinase 6 (GRK6) promoter inhibits binding of C/EBPα, and GRK6 knockdown promotes cell migration and invasion in lung adenocarcinoma cells

doi: 10.1002/2211-5463.12606

Figure Lengend Snippet: GRK6 expression is significantly downregulated in LADC tissues. (A) qPCR results showed that the expression of GRK6 was significantly decreased in LADC tissues compared with that in the adjacent non‐tumor tissues ( P < 0.001, paired Student's t test). (B) TCGA database analysis results showed a decrease in GRK6 expression in LADC tissues compared with that in non‐tumor tissues. The data represent the results from three independent experiments.

Article Snippet: To inhibit the expression of GRK6, siRNA targeting GRK6 was synthesized by GeneChem (Shanghai, China).

Techniques: Expressing

Journal: FEBS Open Bio

Article Title: Hypermethylation of the G protein‐coupled receptor kinase 6 (GRK6) promoter inhibits binding of C/EBPα, and GRK6 knockdown promotes cell migration and invasion in lung adenocarcinoma cells

doi: 10.1002/2211-5463.12606

Figure Lengend Snippet: Downregulation of GRK6 expression in LADC tissues is associated with hypermethylation of the CpG island in the promoter. (A) A schematic map of the GRK6 promoter region indicates the location of the CpG island (blue region). The positions for MSP and BSP primers are also indicated. (B) Representative results of methylation analysis by MSP in LADC and matched adjacent non‐tumor tissues. Case numbers are shown at the top. M, methylated primers; U, unmethylated primers. (C) The methylation level of the GRK6 gene promoter in LADC tissues (Tumor) was higher than that in the matched adjacent non‐tumor tissues (Non‐tumor) in LADC patients (* P < 0.01, paired Student's t test). Data are presented as means ± SD. (D) Correlation analysis between the methylation level of the GRK6 gene promoter and gene expression ( r 2 = 0.35, P < 0.001). (E) Kaplan–Meier survival analysis of LADC patients with unmethylated, partially methylated and hypermethylated GRK6 promoters ( P = 0.027, log‐rank test). The data represent the results from three independent experiments.

Article Snippet: To inhibit the expression of GRK6, siRNA targeting GRK6 was synthesized by GeneChem (Shanghai, China).

Techniques: Expressing, Methylation, Gene Expression

Journal: FEBS Open Bio

Article Title: Hypermethylation of the G protein‐coupled receptor kinase 6 (GRK6) promoter inhibits binding of C/EBPα, and GRK6 knockdown promotes cell migration and invasion in lung adenocarcinoma cells

doi: 10.1002/2211-5463.12606

Figure Lengend Snippet: Clinicopathological characteristics and GRK6 methylation in 54 LADC patients

Article Snippet: To inhibit the expression of GRK6, siRNA targeting GRK6 was synthesized by GeneChem (Shanghai, China).

Techniques: Methylation

Journal: FEBS Open Bio

Article Title: Hypermethylation of the G protein‐coupled receptor kinase 6 (GRK6) promoter inhibits binding of C/EBPα, and GRK6 knockdown promotes cell migration and invasion in lung adenocarcinoma cells

doi: 10.1002/2211-5463.12606

Figure Lengend Snippet: Analysis of the methylation status of the GRK6 gene promoter in LADC tissues and adjacent non‐tumor tissues determined by a BSP assay. (A) A schematic map of CpG islands showing the locations of the 15 CpG sites in the GRK6 gene promoter region. (B) Representative BSP results of methylation in LADC tissues and matched adjacent non‐tumor tissues of case 5 and case 13. Each row represents an individual cloned allele. Each circle represents a CpG site. The black circles show methylated CpG sites, and the white circles show unmethylated CpG sites. The data represent the results from three independent experiments.

Article Snippet: To inhibit the expression of GRK6, siRNA targeting GRK6 was synthesized by GeneChem (Shanghai, China).

Techniques: Methylation, PCR-BSP Assay, Clone Assay

Journal: FEBS Open Bio

Article Title: Hypermethylation of the G protein‐coupled receptor kinase 6 (GRK6) promoter inhibits binding of C/EBPα, and GRK6 knockdown promotes cell migration and invasion in lung adenocarcinoma cells

doi: 10.1002/2211-5463.12606

Figure Lengend Snippet: GRK6 expression is induced by 5‐Aza‐CdR treatment in LADC cell lines. (A) qPCR results showed that the expression of the GRK6 gene in A549 and A427 cells was significantly less than that in 16HBE cells (* P < 0.05, Student's t test). Data are presented as means ± SD. (B) The methylation status of the GRK6 gene promoter was analyzed by BSP in the different cells. (C) GRK6 mRNA expression levels were analyzed by qPCR in the indicated cells treated with 5‐Aza‐CdR (* P < 0.05, Student's t test). Data are presented as means ± SD. The data represent the results from three independent experiments.

Article Snippet: To inhibit the expression of GRK6, siRNA targeting GRK6 was synthesized by GeneChem (Shanghai, China).

Techniques: Expressing, Methylation

Journal: FEBS Open Bio

Article Title: Hypermethylation of the G protein‐coupled receptor kinase 6 (GRK6) promoter inhibits binding of C/EBPα, and GRK6 knockdown promotes cell migration and invasion in lung adenocarcinoma cells

doi: 10.1002/2211-5463.12606

Figure Lengend Snippet: C/EBPα binding to the CpG island of the GRK6 gene promoter. (A) This diagram shows the location of the predicted C/EBPα‐binding sites in the GRK6 gene promoter. The sequences of the predicted binding sites for C/EBPα are listed. (B) A ChIP assay identified the direct binding of C/EBPα to the GRK6 gene promoter. The ChIP‐enriched DNA fragments of the GRK6 gene promoter using IgG and an anti‐C/EBPα antibody were amplified by PCR. Total input was used as a positive control. (C) Sequential deletion analysis identified C/EBPα binding sites in the GRK6 gene promoter region and the relative luciferase activities were determined (* P < 0.05, Student's t test). Data are presented as means ± SD. (D) The binding of C/EBPα with the GRK6 gene promoter in LADC tissues was significantly enhanced compared with that in the corresponding non‐tumor tissues (* P < 0.05, Student's t test). Data are presented as means ± SD. (E, F) The binding strength of C/EBPα to GRK6 gene promoter increased significantly after 5‐Aza‐CdR treatment, compared with cells without 5‐Aza‐CdR treatment (* P < 0.05, Student's t test). Data are presented as means ± SD. The data represent the results from three independent experiments.

Article Snippet: To inhibit the expression of GRK6, siRNA targeting GRK6 was synthesized by GeneChem (Shanghai, China).

Techniques: Binding Assay, Amplification, Positive Control, Luciferase

Journal: FEBS Open Bio

Article Title: Hypermethylation of the G protein‐coupled receptor kinase 6 (GRK6) promoter inhibits binding of C/EBPα, and GRK6 knockdown promotes cell migration and invasion in lung adenocarcinoma cells

doi: 10.1002/2211-5463.12606

Figure Lengend Snippet: C/EBPα is involved in the regulation of GRK6 expression. (A) Western blotting analysis showed that the expression of C/EBPα in the cells transfected with pcDNA3.1‐C/EBPα significantly increased, and enhanced C/EBPα expression significantly upregulated the expression of the GRK6 protein. (* P < 0.05, Student's t test). Data were described as means ± SD. The data represent the results from three independent experiments. (B) The expression of C/EBPα in the cells treated with C/EBPα siRNA was significantly decreased compared with that of cells treated with scrambled siRNA, and inhibition of C/EBPα expression significantly downregulated the expression of the GRK6 protein. (* P < 0.05, Student's t test). Data were described as means ± SD.

Article Snippet: To inhibit the expression of GRK6, siRNA targeting GRK6 was synthesized by GeneChem (Shanghai, China).

Techniques: Expressing, Western Blot, Transfection, Inhibition

Journal: FEBS Open Bio

Article Title: Hypermethylation of the G protein‐coupled receptor kinase 6 (GRK6) promoter inhibits binding of C/EBPα, and GRK6 knockdown promotes cell migration and invasion in lung adenocarcinoma cells

doi: 10.1002/2211-5463.12606

Figure Lengend Snippet: The effects of GRK6 on cell migration and invasion. (A) After transfection with siRNA or pcDNA3.1‐GRK6, the expression of the GRK6 protein decreased or increased significantly, respectively (* P < 0.05, Student's t test). Data are presented as means ± SD. (B) Upregulation of GRK6 significantly inhibited cell migration and invasion (* P < 0.05, Student's t test). Data are presented as means ± SD. (C) Downregulation of GRK6 significantly promoted cell migration and invasion (* P < 0.05, Student's t test). Original magnification: ×200. Data are presented as means ± SD. The data represent the results from three independent experiments.

Article Snippet: To inhibit the expression of GRK6, siRNA targeting GRK6 was synthesized by GeneChem (Shanghai, China).

Techniques: Migration, Transfection, Expressing

Journal: FEBS Open Bio

Article Title: Hypermethylation of the G protein‐coupled receptor kinase 6 (GRK6) promoter inhibits binding of C/EBPα, and GRK6 knockdown promotes cell migration and invasion in lung adenocarcinoma cells

doi: 10.1002/2211-5463.12606

Figure Lengend Snippet: GRK6 affects EMT phenotypes and MMP expression. (A) GRK6 overexpression significantly increased E‐cadherin expression but inhibited the expression of vimentin (* P < 0.05, Student's t test). Data are presented as means ± SD. (B) GRK6 knockdown reversed all of these changes (* P < 0.05, Student's t test). Data are presented as means ± SD. (C) Upregulation of GRK6 significantly suppressed the expression of MMP2 and MMP7 proteins (* P < 0.05, Student's t test). Data are presented as means ± SD. The data represent the results from three independent experiments.

Article Snippet: To inhibit the expression of GRK6, siRNA targeting GRK6 was synthesized by GeneChem (Shanghai, China).

Techniques: Expressing, Over Expression, Knockdown