The Transcriptional Regulatory Study Of C-1888T Single Nucleotide Polymorphism (SNP) Site In Promoter Region Of PLUNC Gene With The Susceptibility To Nasopharyngeal Carcinoma

BACKGROUND & OBJECTIVENasopharyngeal carcinoma (NPC) was a malignant tumor with high incidence in Southern China and Southeastern Asia. Epstein Barr virus infection, tumor promotion and/or carcinogens all played an important role in NPC tumorigenesis. As the other tumor, NPC carcinogenesis was a multi-cause and multi-step process. To investigate the gene expression changes in NPC carcinogenesis, Yao KT and He ZW compared the differential gene expression profiling between human normal nasopharyngeal and NPC tissue by high density cDNA microarray, then cloned a complete sequence of EST with relatively special expression in human nasopharyngeal and trachea tissue. This new candidate tumor suppressor gene was named YH1 gene with the GenBank accession number AF158745. Subsequently, other research groups cloned an new gene which named PLUNC(palate, lung and nasal epithelium clone, PLUNC) from the nasopharyngeal epithelium of mouse embryonic, adult lung, respiratory tract and nasopharynx.YH1 was highly homologous to the PLUNC gene, and remarkably conserved with other species including pig, cow and rat, so YH1 has been cataloged to PLUNC family. According to the literatures, single nucleotide polymorphisms (SNPs) in coding, flanking and regulating regions of genes were closely related to some diseases and may change the gene’s function. Nine SNPs have been found from these regions of PLUNC gene in our group’s previous study. Two promoter SNPs,C-1888T and C-2128T, showed significant association with susceptibility to NPC(OR=2.8-3.3, P<0.001), haplotypes based on the polymorphisms indicated that individuals with haplotype C-C had significantly increased the susceptibility to NPC(OR1.86,95% CI=1.34-2.56,P=0.00016).The luciferase activity decreased about 64.67% after 1888T→C base variation, the difference was statistically significant. All of these results suggested that C-1888T polymorphism may affect the regulation of gene’s transcription and relate to NPC susceptibility/risk.Based on the above background, it was necessary to study in-depth and validate the reliability of previous results; meanwhile, to verify whether the polymorphism contribute to the transcriptional regulation of the gene and the susceptibility to nasopharyngeal carcinoma/risk.METHODS:1、The methods of PCR-sequence and PCR-RFLP were performed to screen three genotypes from NPC cell lines(CNE1、CNE2、SUNE1、5-8F、6-10B、C666-1) and primary culture of the epithelial cell from human bioptic specimens of NPC.2、Total RNA were extracted from the above NPC cell lines, then reversed transcription into cDNA. The expressions of PLUNC mRNA were detected by the method of quantitative real-time PCR, and One-Way ANOVA of SPSS13.0 was applicted to analyze the results.3、Transcription factors which were more likely combining to C-1888T SNP site were analyzed by p-match(www.gene-regulation.com/pub/programs.html#pmatch), Consite(http://mordor.cgb.ki.se/cgi-bin/CONSITE/consite) and other software. The probes and mutant probes were designed according to the binding site matrix information and transcription factors binding core sequences given by TRANSFAC database.4、The nucleic protein were extracted from cells with CC/TT genotypes and detected concentration. Then, nucleic protein was combined to the labelled probes with unlabelled specific-competitive and unspecific-competitive oligonucleotides. A specific band was observed in the gel.5、Chromatin immunoprecipitation(ChIP) and PCR were further performed to confirm the binding of EVI1 to PLUNC promoter region of CC genotype NPC cell.RESULTS1、NPC cell lines 5-8F、6-10B、CNE1、CNE2 were definited CT genotype, SUNE1 and C666-1 were definited CC and TT genotype, respectively. The PCR-sequence results were coincide with PCR-RFLP. Primary cultured cells which belong to CT genotype grew well, but CC/TT genotypes were in poor status or even death and could not meet the need for subsequent experiments.2、The expressions of different cell lines were statistically significant(F=33.844, P=0.000), there was also a significantly low-expressed of PLUNC mRNA in the CC genotype compared with that in the TT genotype (P=0.000).3、Class to the method of bioinformatical, transcription factors XFD3 or EVI1 had a highly possibility to combine with PLUNC gene(scores 0.96 and 1.00 respectively) when the C-1888T SNP site was A or G. The core sequences of XFD3 and EVI1 were TTGGTCAACAAGAT and CGACAAGATAA respectively. Follow-up experiments confirmed that the forecasting results were accurate and reliable. 4、The region in promoter of PLUNC gene were validated to allow binding of XFD3 or EVI1 when SNP site was A or G by EMSA analysis. The specific band was unaffected by mutant probe, while no specific band was observed with unlabelled probe.5、DNA-protein-antibody complexes were precipitated after CC genotype cells were cross-linked with formaldehyde and incubated with EVI1 antibody. The ChIP results showed that Anti-RNA PolymeraseⅡChIP DNA,EVI1 antibody ChIP DNA and Input DNA were determined to have the-336 to-144bp region of PLUNC promoter after PCR amplification except for the negative control IgG ChIP DNA.CONCLUSION1、The C-1888T SNP site in promoter region of PLUNC was also found to exist in NPC cell lines, so the cells could be used for related researches in vitro.2、The expression of CC genotype was lower than that of TT genotype with significant difference(P=0.000), and we had shown in previous study that 1888C promoter activity was significantly lower than PLUNC based promoter 1888T activity. All of them indicated that the individual was more susceptible to CC genotype may down-regulate the expression of PLUNC gene and thus affected the susceptibility to NPC.3、Bioinformatics was one of the necessary technology to predict transcription factors binding site. The predicted results were consistent with later experiment, which confirmed the accuracy of forecasts.4、EMSA experiments preliminarily indicated that XFD3 and EVI1 may regulates the activity of PLUNC gene, which resulted in susceptibility/risk to NPC.5、ChIP experiments further confirmed that EVI1 could bind to the region in PLUNC gene promoter and regulate the activity of PLUNC gene.