title: The NFKB1 polymorphism (rs4648068) is associated with the cell proliferation and motility in gastric cancer.
abstract: BACKGROUND
  We have demonstrated previously that NFKB1 single nucleotide polymorphism (SNP) rs4648068 GG homozygote was associated with the increased risk of gastric cancer in Chinese Han population. In this study, we constructed the recombinant plasmid pGL3-AA, pGL3-GG, pGL3-AA-NFKB and pGL3-GG-NFKB to investigate the function of rs4648068 by cell biology experiments.
  
  
  METHODS
  Quantitative real-time PCR was used to detect NFKB1 SNP rs4648068 genotype in the patients with gastric cancer. Anti-NF-κB1 p50 polyclonal antibodies were used for immunohistochemical analysis of the tissue specimens. The subsection of NFKB1 containing the promoter site and adjacent three consecutive exons were obtained by PCR technique and subcloned into the vector pGL3-Basic. Dual-Luciferase reporter assay was used to detect the transcriptional activity of the constructed promoter. Effects of transcription factor NFKB1 on C/EBPβ expression were determined by chromatin immunoprecipitation and Western analysis. Furthermore, proliferation and invasion ability of the transduced cell were also measured and compared.
  
  
  RESULTS
  Intensive staining for p50 expression was observed in the tissues of GG genotype patients, compared with those of GA group and AA genotype patients. The transcriptional activity of rs4648068 (A > G) by dual-Luciferase reporter assay suggested that the luciferase activity of homozygote group (pGL3-GG) was greater than that of the control (pGL3-AA), especially at the stimulation of LPS. We found that the luciferase activity was also influenced by pGL3-GG levels. The effects of NFKB1 rs4648068 were enhanced by rs4648065 on the transduced cells. The interaction between NFKB1 promoter nucleotide sequence and C/EBPβ was regulated by the functional SNP rs4648068 in SGC-7901 cells. Our data indicated that the transduction of pGL3 expression plasmid pGL3-GG-NFKB improved the proliferation and motility of gastric cancer cells. Correspondingly, the homozygote GG of SNP rs4648068 strengthened the transcriptional activity of NFKB1 and influenced the cell biological activity.
  
  
  CONCLUSIONS
  The transcriptional activity of NFKB1 was associated with SNP rs4648068, and this functional SNP site has the important effects on cell proliferation and motility.
authors.affiliation: Department of Clinical Laboratory, Shanghai Cancer Center, Fudan University, Shanghai, 200032, China. cygl_fudan@126.com.|Department of Oncology, Shanghai Medical College, Fudan University, Shanghai, 200032, China. cygl_fudan@126.com.
authors.forename: Ying
authors.initials: Y
authors.lastname: Chen
authors.affiliation: Department of Clinical Laboratory, Shanghai Cancer Center, Fudan University, Shanghai, 200032, China. lurenquan66@hotmail.com.|Department of Oncology, Shanghai Medical College, Fudan University, Shanghai, 200032, China. lurenquan66@hotmail.com.
authors.forename: Renquan
authors.initials: R
authors.lastname: Lu
authors.affiliation: Department of Clinical Laboratory, Shanghai Cancer Center, Fudan University, Shanghai, 200032, China. zh19841207xx@163.com.
authors.forename: Hui
authors.initials: H
authors.lastname: Zheng
authors.affiliation: Department of Clinical Laboratory, Shanghai Cancer Center, Fudan University, Shanghai, 200032, China. ljerry19890505@hotmail.com.
authors.forename: Ran
authors.initials: R
authors.lastname: Xiao
authors.affiliation: Department of Clinical Laboratory, Shanghai Cancer Center, Fudan University, Shanghai, 200032, China. fjj83996@163.com.
authors.forename: Jingjing
authors.initials: J
authors.lastname: Feng
authors.affiliation: Department of Oncology, Shanghai Medical College, Fudan University, Shanghai, 200032, China. HonglingWang123@163.com.
authors.forename: Hongling
authors.initials: H
authors.lastname: Wang
authors.affiliation: Department of Clinical Laboratory, Shanghai Cancer Center, Fudan University, Shanghai, 200032, China. gxcy2001@hotmail.com.
authors.forename: Xiang
authors.initials: X
authors.lastname: Gao
authors.affiliation: Department of Clinical Laboratory, Shanghai Cancer Center, Fudan University, Shanghai, 200032, China. 1251487447@qq.com.|Department of Oncology, Shanghai Medical College, Fudan University, Shanghai, 200032, China. 1251487447@qq.com.
authors.forename: Lin
authors.initials: L
authors.lastname: Guo
mesh_terms: D000230:Adenocarcinoma / Q000737:chemistry / Q000235:genetics*; D022762:CCAAT-Enhancer-Binding Proteins / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000235:genetics*; D049109:Cell Proliferation / Q000235:genetics*; D004274:DNA, Recombinant; D015870:Gene Expression / Q000187:drug effects; D017930:Genes, Reporter; D005822:Genetic Vectors; D005838:Genotype; D006801:Humans; D008070:Lipopolysaccharides / Q000494:pharmacology; D008156:Luciferases / Q000235:genetics; D052002:NF-kappa B p50 Subunit / Q000032:analysis / Q000235:genetics*; D010957:Plasmids; D020641:Polymorphism, Single Nucleotide; D011401:Promoter Regions, Genetic; D013274:Stomach Neoplasms / Q000737:chemistry / Q000235:genetics*; D014158:Transcription, Genetic; D014161:Transduction, Genetic
chemical_list: D022762:CCAAT-Enhancer-Binding Proteins; D004274:DNA, Recombinant; D008070:Lipopolysaccharides; D052002:NF-kappa B p50 Subunit; C489548:NFKB1 protein, human; D008156:Luciferases