A Preliminary Study on Transcriptional Regulation of SNP Site C-1888T in the Promoter Region of Human PLUNC Gene and Nasopharyngeal Carcinoma Susceptibility

Abstract

Purpose: The transcriptional regulatory factors binding to the polymorphic site C-1888T in the promoter region of the palate, lung, and nasal epithelium clone (PLUNC) gene were identified to investigate whether the C-1888T polymorphic site affects the transcriptional regulation and function of PLUNC gene. Materials and Methods: Three genotypes of C-1888T polymorphic locus were screened from established nasopharyngeal carcinoma (NPC) cells, and the mRNA expression levels of PLUNC gene in different genotypes were detected. The respective transcription factors that were more likely to bind with A or G in SNP were predicted by biological information and preliminarily verified in vitro by gel electrophoresis migration rate analysis. Ulteriorly, the NPC cell lines were analyzed through chromatin immunoprecipitation combined with PCR amplification to confirm that the transcription factors could bind to the PLUNC gene promoter. Results: The cell lines 5-8F, 6-10B, CNE1, and CNE2 were heterozygous CT type, SUNE1 was homozygous CC type, and C666-1 was homozygous TT type. The expression of PLUNC gene was significantly different among all cell lines (F = 33.844, p < 0.001), and the gene expression level of CC type was significantly lower than TT type (p < 0.001). Gel electrophoresis mobility analysis confirmed that the transcription factors XFD3 and EVI1 could bind to the PLUNC gene promoter when the SNP was A and G, respectively. PCR amplification combined with chromatin immunoprecipitation showed that EVI1 could bind to the DNA fragment of the promoter region of PLUNC gene in SUNE1 NPC cells. Conclusion: The transcription factors XFD3 and EVI1 may be involved in the transcriptional regulation of PLUNC gene, and EVI1 can bind to the promoter region of PLUNC gene in SUNE1 NPC cells, thus associated with the susceptibility/risk of NPC.

Keywords: PLUNC genes; nasopharyngeal carcinoma; single-nucleotide polymorphism; transcriptional regulation.

Figure 1

(a) PCR products were digested by FbaI enzyme. (b) Sequencing results. (A) represents 1 in (a) (homozygous CC type), (B) represents 2, 3, and 4 in (a) (heterozygous CT type), and (C) represents 5 in (a) (homozygous TT type).

Figure 2

Prediction transcription factor binding site of C-1888T.

Figure 3

Matrix of transcription factor binding site.

Figure 4

The specific binding of SNP site C-1888T in the promoter region of PLUNC gene to transcription.

Figure 5

PCR analysis of EVI1 binding with PLUNC gene promoter in SUNE1 NPC cell line. M: marker, 1: input DNA, 2: IgG ChIP DNA, 3: EVI1 antibody ChIP DNA, 4: anti-RNA polymerase II ChIP DNA.