Colorectal cancer-associated SNP rs17042479 is involved in the regulation of NAF1 promoter activity

Abstract

A novel risk locus at 4q32.2, located between the Nuclear Assembly Factor 1 (NAF1) and Follistatin Like 5 (FSTL5) genes, was associated with increased risk of developing colorectal cancer (CRC), with SNP rs17042479 being the most associated. However, the link between CRC development and the risk locus at 4q32.2 is unknown. We investigated the promoter activity of NAF1 and FSTL5 and analyzed the risk locus at 4q32.2 as gene regulatory region. Our results showed that the activity of the FSTL5 promoter was low compared to the NAF1 promoter. Analyses of the NAF1 promoter in conjunction with the region containing the risk locus at 4q32.2 showed that the region functions as gene regulatory region with repressor activity on NAF1 promoter activity. The SNP rs17042479(G) increased the repressor effect of the region. CRC patients' biopsies were genotyped for SNP rs17042479(A/G), and NAF1 expression profiles were examined. We found an association between SNP rs17042479(G), cancer stage and tumor location. Additionally, patients with SNP rs17042479(G) showed lower NAF1 expression in comparison to patients with SNP rs17042479(A) in tumor tissue and the NAF1 expression in tumor tissue was lower compared to healthy tissue. The results in the study imply that reduced NAF1 expression in the tumor contribute to a more aggressive phenotype. Furthermore, this study suggests that the SNP rs17042479(G) change the expression of NAF1 and thereby increases the risk of developing CRC.

Fig 1

(a) Location of the risk locus at 4q32.2. CAGE analysis [14] of the region containing the two genes NAF1 and FSTL5 and the risk locus at 4q32.2, shown in yellow, are presented in the top [16]. The bottom presents a zoom in of the risk locus at 4q32.2, that shows the 6 SNPs found in Schmit et al., highlighted in blue. From left: rs17042479 (A/G), rs79783178 (-/AT), rs35509282 (A/T), rs11736440 (A/G), rs9998942 (C/T) and rs57336275 (C/T) (b) Plasmid map of the construct used in the promoter reporter gene assay of promoter activity (c) Promoter reporter assay. Promoter activity of FSTL5 (FSTL5pro) and NAF1 (NAF1pro) in colon cancer cell lines. From left to right: Caco2 cells, DLD-1 cells and SW480 cells. Luciferase activities were corrected for transfection efficiency and normalized to the activity of the pGL4.10 without promoter (pGL4.10). N = 4. Statistical significance was determined using one-way ANOVA. (d) Plasmid map of the construct used in the promoter reporter gene assay of the gene regulatory region (e) Promoter reporter assay. The luciferase activities were corrected for transfection efficiency and normalized according to the expression of the NAF1 promoter (NAF1pro). From left to right: Caco2 cells, DLD-1 cells and SW480 cells. NAF1pro + SNP rs17042479(A) is the construct with the NAF1 promoter and the reference allele of SNP rs17042479. NAF1pro + SNP rs17042479(G) is the construct with the NAF1 promoter and the risk allele of SNP rs17042479. N = 4. Statistical significance was determined using one-way ANOVA.

Fig 2

(a) Barplot of SNP rs17042479(G) distribution in cancer stages. The analysis was examined by Chi2-test and Fisher’s exact test; p-value = 0.02. (b) Barplot of SNP rs17042479(G) distribution in tumor location. The analysis was examined by Chi2-tests and Fisher’s exact test; p-value = 0.03.

Fig 3

(a) Boxplot of NAF1 expression in respectively healthy and tumor tissue. The patients with tumor content under 50% are not included in the analysis. n = 121. The log transformed data was examined by paired t-test; p-value = 0.012. (b) Boxplot of the NAF1 expression in healthy tissue and the presence of SNP rs17042479(G). n = 43 for patients with the risk allele and n = 155 for patients with the reference allele. The analysis was examined by unpaired t-test; p-value = 0.28. (c) Boxplot of the NAF1 expression in tumor tissue and the presence of SNP rs17042479. The patients with tumor content under 50% are not included in the analysis. n = 22 for patients with the alternative allele, and n = 99 for patients with the reference allele. The analysis was examined by unpaired t-test; p-value = 0.05.