Identification and Characterization of Putative Nasopharyngeal Carcinoma-susceptible gene around Human Leukocyte Antigen C Locus

• Main Authors: Tsu-Hsin Hsueh, 薛祖欣
• Other Authors: Cheng-Chan Lu
• Format: Others
• Language: en_US
• Published: 2008
• Online Access: http://ndltd.ncl.edu.tw/handle/06324151007557296637

碩士 === 國立成功大學 === 分子醫學研究所 === 96 === Nasopharyngeal carcinoma (NPC) is a squamous cell carcinoma that is highly prevalent in southern Chinese, including people living in Taiwan. Numerous pieces of evidence suggest that genetic factors provide background susceptibility on which EBV infection and environmental factors may interact with each other and lead to the development of NPC. In a previous study, using HLA gene as linkage disequilibrium mapping tool and microsatellite marker for analysis, we have further identified a second NPC-susceptible locus localized within HLA-C region. Further study using microsatellite markers spanning 780 kb across the HLA-B and -C loci have narrowed down this NPC-susceptibility locus to within 54 kb. This study was divided into two major parts. Part I: To identify whether any DNA sequence variation is associated with genetic susceptibility to NPC within this 54 kb segment of putative NPC-susceptibility region. By DNA sequencing and genotyping study, we have first identified two genetic variations significantly associated with NPC among a number of single nucleotide polymorphisms (SNPs) found within this region. One is HCR-SNP 21 (g.11648 C>T, rs3132539) (p=0.03, OR=0.49, T carrier mode), another is SC1-SNP 8 (g.2335 G>A, rs2073721) (p=0.01, OR=2.19, G carrier mode). Part II: Functional study of SC1-SNP 8. SC1-SNP 8 caused an amino acid substitution at position 211 (V211M). Literature searches indicate that SC1 may play a role in cell cycle regulation. The genetic analysis of a number of NPC cell lines indicated that they all expressed G/G homozygote, and G allele is conserved in various mammalian species. To examine if this amino acid change may have any effect on the cell cycle distribution, wild type and variant expression vectors (pHA-SC1 variants/IRES2-EGFP) were transfected into mouse NIH/3T3 fibroblasts or NPC cell lines. In cell cycle distribution analysis, we found that the distinct cell cycle distribution of SC1 variants at early S phase of cell cycle. To investigate if the amino acid replacement may have any effect on the subcellular localization of protein expression, wild type and variants of SC1 and EGFP fusion protein-expressing plasmids were also constructed. The confocal microscope study revealed that SC1 V211-EGFP fusion protein was expressed in both nucleus and cytoplasm, but SC1 M211-EGFP fusion protein was predominantly expressed in cytoplasm. Moreover, the luciferase reporter assay indicated that NLMP1 may upregulate the promoter activity of SC1 gene. These data suggested that NLMP1 may regulate the SC1 gene expression at the transcription level, and the differential nucleocytoplasmic distribution of SC1 variants may cause the distinct cell cycle distribution at early S phase of cell cycle. Furthermore, we found that distinct inducing effects of SC1 variants on the promoter activity of HCGIV-06 (another putative NPC-susceptible gene). Finally, an unexpected we identified two novel alternative splice variants of SC1 from RT-PCR analysis of SC1 cDNA. The biological function of these two variants is currently unknown. The identification of NPC susceptible gene may shed light on how they may contribute to the development of NPC.