Figure 6. Effects of NKX6.3 on GKN1 expression.

A. Putative promoter activity was characterized between −5 kb and +1 kb relative to the transcription start site (TSS) of GKN1 by ChIP and QPCR. Binding activity of NKX6.3 was detected in the GKN1 promoter region. Luciferase activity of AGS^NKX6.3 cells transfected with plasmids with full-length (−5 to +1 kb) or deletions of the GKN1 promoter containing TSS designated 0 kb, then cultured for 24 h. Luciferase activity showed that NKX6.3 occupancy was 20-fold enriched at the P4 region (positions −2 to +1 kb) compared to the control. Normalized luciferase activity values for each construct (N = 3, *<0.05, t-test) are represented as mean ± SD. B. Five putative NKX6.3 binding motifs were found at the P4 promoter region. Luciferase activity analysis of 5′-deletion constructs at the P4 promoter region showed a significant decrease in promoter activity. Further deletion of the 5′ binding motifs resulted in a progressive loss of activity, indicating that NKX6.3 binding motifs at the P4 construct are required for the GKN1 transcription. C. NKX6.3 induced GKN1 mRNA and protein expression in AGS^NKX6.3 and MKN1^NKX6.3 cells. D. There was positive correlation between NKX6.3 and GKN1 expression in 55 non-cancerous gastric mucosa and large cohort of gastric cancer patients (NCBI GEO database, accession numbers GSE27342).