The Preliminary Study Of Functional C-1888t Single Nucleotide Polymorphism (snp) Site In Promoter Region Of Hplunc Gene In Nasopharyngeal Carcinoma

BACKGROUND & OBJECTIVENasopharyngeal carcinoma (NPC) may be a multifactorial, multistep and multipathway progression which concerned with the accumulation of genetic variation. Now, it was associated with Epstein-Barr virus (EBV) infection, environmental factors and genetic susceptibility. The statistical analysis showed that about 10% NPC Patients had a family history, which suggested genetic susceptibility might play an important role in the pathogenesis of NPC.A lot of research methods, such as the epidemiologic investigation, the familial aggregation studies, and the genetic model analysis indicated that NPC is a multi-gene hereditary disease, concerned with a number of oncogenes and/or suppressor genes. PLUNC (palate, lung, and nasal epithelial clone gene) is the one of that genes, located in the human chromosome segment 20q11.2. The full length of PLUNC is 1096bp and can be delineated into 9 exons and 8 introns. It was reported that PLUNC gene may be regarded as a candidate suppressor of NPC due to its abnormal expression in NPC development.According to the literature, gene polymorphisms of promoter region are closely related to some diseases. Our previous research has also found the association of functional single nucleotide polymorphisms (SNPs) with nasopharyngeal carcinoma in a Chinese cross-sectional study. On the basis of large sample statistics, we demonstrated that C-1888T SNPs in the promoter region of PLUNC gene were significantly associated with NPC. Using PCR direct sequencing, we not only found the two above promoter SNPs were closely related to NPC susceptibility in Guangdong population, but also indicated the distribution of haplotypes, which were constructed based on the polymorphism C-1888T, was significantly different between NPC patients and controls, suggesting that the individuals with haplotype C-C had significantly increased susceptibility to NPC. Therefore, these data suggested that PLUNC gene might act as a genetic risk factor for NPC in Cantonese-speaking Chinese patients and that a risk allele could significantly affect the susceptibility to NPC in the Chinese population by means of disturbing the spatial/temporal expression of PLUNC GENE (Some of this results have been published). However, the exact mechanism by which PLUNC gene may affect the susceptibility of NPC is not as yet clear.As is mentioned above, we intend to study the influence of the C-1888T SNP site on the PLUNC gene expression in transcription level, to explore the reasons for the susceptibility differences to nasopharyngeal carcinoma of different genotypes and to reveal a possible factor of NPC. So we have made some researches to elucidate the characteristics and regulatory mechanisms of NPC as follows:METHODS:1,In order to gain the 1888 SNP site and other SNPs nesrby and find all the genetypes, DNAs which originated from various cells and peripheral blood, were examined by PCR-sequence and PCR-RFLP.2,The about 280bp DNA fragments of PLUNC gene promoter region including the 1888 SNP site were obtained by polymerase chain reaction ( PCR) based on the results of CHAPTER I with the CC/TT genotypes, which were cloned to pMD18- T Simple vectors and pGL3-enhancer luciferase reporter gene vector successively. Then, the two recombinant plasmids (pGL3-Enhancer-TT and pGL3-Enhancer-CC) containing different haplotypes DNA of 1888 SNP site were obtained, whose sequences completely matched with the theoretical prediction demonstrated by sequencing. After transfecting transiently in nasopharyngeal cancer cell line HONE1, the relative luciferase activity (RLA) was measured. All the data were statistically analysed by one-way ANOVA and Dunnett's T3 of SPSS13.0.3,Synthesizing four-type DNA fragments which are 31 bp with G/ C/ A/ T on the 1888 SNPsite separately. Then, the DNAs were labeled with Biotin-11-dUTP by terminal transferase as the probes. Meanwhile, the nuclear extract was prepared from CNE2 cells and DNA—protein interactions were examined by electrophoretic mobility shift assay(EMSA).RESULTS1,Sequencing of the -437 bp～+87 bp region of the PLUNC gene promoter in the seven nasopharyngeal carcinoma cell lines and the blood samples from NPC patients has found all the three gene types on 1888 SNP site and revealed other three mutant sites comparing with the public sequences from NCBI. The 2128 SNP site have been proved to be functional polymorphisms. The other two (N1 and N2) have not been identified in our previous research. After searching the NCBI datebase (http://www.ncbi.nlm.nih.gov/) , the SNP of N2 site in the PLUNC gene are represented with the following GenBank accession numbers: rs11907039, but the mutation of N1 site have not been described previously in clinical samples and their functional role is uncertain.2,Sequencing confirmed that the recombinant plasmids containing C-1888T SNP site were obtained. Comparing the luciferase activity of the two recombinant plasmids (pGL3-Enhancer-TT and pGL3-Enhancer-CC), the difference was statistically significant (P <0.05) analysed by one-way ANOVA and Dunnett's T3 of SPSS13.0. And the luciferase activity decreased significantly by about 64.67% after 1888 Tâ†'C base variation.3,EMSA results showed that: In the competition study with labeled probe of G/C type with nuclear protein, negative control group and competitive inhibition group, the formation of A/T type complex was inhibited, indicating that the binding of the A/T type probe to the CNE2 nuclear protein was specific.CONCLUSION1,The results of Sequencing laid a solid foundation for testing functional transcription activity of different genotypes in PLUNC gene promoter region. And it indicated that the standard nasopharyngeal carcinoma cell lines in vitro also display the mutation phenomenon on 1888 SNP site which were reported in blood samples from NPC patients. The newly discovered mutation point N1 may be a new SNP site which needs to be further investigated in larger studies.2,The detection of luciferase activity reconfirmed that C-1888T may be functional mutation point in the promoter region of the PLUNC gene, after large sample statistics. And CC-genetype on 1888 SNP point may regulate the expression of PLUNC gene and thus affect the susceptibility to nasopharyngeal carcinoma.3,EMSA experiments, which indicated that the binding capacity of abeled probe of A/T type with nuclear protein was obviously stronger than G/C type, further proved that there are differences between different genotypes on 1888 SNP site of PLUNC gene promoter region in transcription level. So we can conclude that the reason why the susceptibility to nasopharyngeal carcinoma is different may be the decline of the transcription activity when the genetype has changed on 1888 SNP site.In short, we detected the transcription activity of different genotypes on 1888 SNP site of PLUNC gene promoter region by the relative luciferase activity (RLA) examination and electrophoretic mobility shift assay (EMSA). The results suggest that transcriptional activity has declined markedly when the genetype has changed from TT to CC on 1888 SNP site. Our previous research proved that the individuals with haplotype C-C on the 1888 SNP site in the promoter region of the PLUNC gene had significantly increased susceptibility to NPC. Then whether it concerns with the decline of the transcription activity or not needs our in-depth study as the specific regulatory mechanism has not yet be completely clear.