| AIMS: VDR, the key transcriptional regulator, regulate a series of target gene transcription in the downstream, as if CYP3A4, MDR1. It can influence target genes transcription and expression with itself change. 1, 25-(OH)2D3 is an essential vitamin that belongs to steroid hormone, the principal recognized biological effect of 1, 25-(OH)2D3, through its combined actions on classical endocrine target tissues including intestine, bone, and kidney, is to maintain calcium/phosphate ion homeostasis, regulation cell growth or differentiation; liganding of the vitamin D receptor(VDR) withl, 25-(OH)2D3 mediates the effects of the 1, 25-dihydroxyvitamin D3 to alter a series of target gene transcription of downstream. In addition, VDR may play an important role in drug metabolism and therapeutic effect; and its abnormity will change the quantity and quality of protein, causing alteration of pharmacokinetics, pharmacodynamics, even clinical diseases and so on. In present study, we delineated the characterization of a VDR Fokl to explore the association of the polymorphism of VitaminD receptor(VDR) gene in the start codon with the risk of colorectal tumor and if this variant could alter its target gene transcription and expression of CYP3A4 in order to study the possible molecular mechanism for colorectal tumor.Methods: The VDR genotypes were determined by polymerase-chain-reaction-restriction-fragment-length polymorphism (endonuclease FokI) in 69 patients with colorectal tumor and 218 normal controls. Then we cloned the cDNA sequence of human vitamin D receptor(VDR) gene (wild type and mutant) and establish their eukaryotic expression vectors. Moreover, we utilize cell transfection technique and dual-luciferase report gene analytical system in vitro to investigate the Fokl mutant of VDR and its comparison with wild type of VDR as well as the effect of the 1, 25-dihydroxyvitamin D3 on transcription of CYP3A4 in cultured COS-7 cell. Results: The frequencies distribution of FokI alleles in this cohort all followed the Hardy-Weinberg equilibrium. Our study showed that the F allelic frequency was 61.4％in Chinese healthy volunteers, a significant difference existed between South China(74.3％), Japanese(41.7％) and colorectal tumor patients.(49％) (P＜0.05). Between normal controls and patients, The distribution of this genotype FF, Ff, ff frequencey is 39.3％, 44.2％, 16.5％vs 29％, 40％, 31％respecively. The frequency of F allele and FF genotype in patients are lower than controls and this three genotype frequency had statistically significant diferences (P＜0.05). We found that compared with individuals carrying the FF genotype, those with Ff genotype had a two fold in risk of colorectal cancer(OR=2.00; 95％CI:1.01-3.96;) and those with the ff genotype, 2.5 fold (OR=2.508; 95％CI:1.21-5.18)in risk(P for trend=0.01). On the base of successfully constructing the mutant and wild type of pcDNA3.1(-)B-myc/his hVDR, in transiently transfected COS-7 cells, the CYP3A4 luciferase activity of three concentrations(1, 10, 100nmol/L)was increasing respectively after 1, 25(OH)2VD3 was added to cells for 24 hours,. There were significant difference between 1, 25(OH)2VD3 group and the vehicle control group, and the luciferase activity from mutant VDR constructs resulted a little greater than wild VDR constructs, but there was no significant differences between the two VDR forms (P＞0.05).Conclusions: The polymorphism of Vitamin D receptor(VDR) gene in the start condon is associated with the risk of colorectal tumor. The plasmids for encoding hVDR cDNA were successfully cloned from liver tissure of Chinese and their eukaryotic expression vector(wild type and mutant) pcDNA3.1(-)B-myc/his hVDR, were successfully constructed. Results in this study showed whatever any genotype of VDR constructs, 1, 25(OH)2VD3 had a concentration-dependent induction effect on CYP3A4mRNA, but we cannot detect significant differences in transactivation activity on CYP3A4 between wild and FokI mutant VDR constructs. |
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