Single Nucleotide Polymorphism And Expression Regulation Of Vitamin D Receptor Gene In Idiopathic Hypercalciuria

Objective To establish a colony of geneticlly hypercalciuria rats model which can stablely descending and explore the pathogenetic role in intestinal calcium hyperabsorption with the rats model, and obtain more information concerning single nucleotide polymorphism (SNP) of Vitamin D receptor gene of GHS rats in Idiopathic hypercalciuria.Methods 40 male and 40 female adult Sprague-Dawley rats were screned for hypercalciuria. The rats were placed in individual metabolic cages. Two successsve 24h urine collections were then obtained to measure the urine calcium excretion. The three male and three female rats used to breed the next generation. A similar protocol was used to select the two to three hypercalciuric males and three to four hypercalciuric females for inbreeding of subsequent generations. Then 42 GHS rats and 24 normal control rats took part in this study. Genomic DNA was extracted from peripheral leukocytes isolated from EDTA anticoagulated blood by standard method with phenol and chloroform. All the gene from 5' -regulation region of VDR were amplified by PCR. After purified, the PCR products were sequenced.Results Urine calcium excretion in 12/15 males and 9/16 females were significantly above the mean of the controls. The content of serum calcium, phosphorus and l,25(OH)₂D₃ were similar(P>0.05). Six polymorphism sites were found in GHS rats. There were significant differences in two of SNPs between GHS rats and normal controls.Conclusion Genetic hypercalciuric rats model were established succesfully and they are similar to human idopathic hypercalciuria , which may be familial and is thought to be genetic in origin. So the model is suitable for the study on the mechanism of idiopathic hypercalciuria. These findings suggest that the single nucleotide polymorphism in 5'-regulation region of the VDR gene may play a significant role in mechanism of Idiopathic hypercalciuria. Objective To evaluate VDR gene promoter on gene transcription, we constructluciferase reporter plasmid pGL3-GHS-Luc and pGL3-N-Luc.Methods The VDR DNA of GHS rats and normal control rats were used as templatesin the polymerase chain reaction to amplify their upstream regions from thetranscription initiation sites. The PCR products were directly cloned intothe luciferase reporter vector pGL3 - Basic. pGL3-GHS-Luc and pGL3-N-Luc wereconfirmed by constriction analyses.Results Restriction enzymes digestion and nucleotide sequencing confirmedthat the recombinant plasmids were correct without base mutation and deletion.There were only two differents of single nucleotide between pGL3-GHS-Luc andpGL3-N-Luc.Conclusion Rat VDR promoter luciferase reporter gene plasmids weresuccessfully constructed. The construction of VDR recombinant plasmid lay afoundation of the analysis of promoter activities and make an important basisfor studying transcrip tional regulation mechanisms of VDR gene. Objective To study the impact of VDR 5' flanking promotor of rats on theexpression of luciferase in Hela cell, and evaluate in vitro regulation of VDRpromoter on gene transcription.Methods pGL3-GIS, pGL3-N and pGL3-Basic were transfected into Hela cells bylipofectin and selected by G418 respectivly, after amplifiction of thepositive cell clones, expression of luciferase was detected andquantitatively analysised by digital image system.Results Reporter gene plasmids under the direction of the VDR 5' flankingpromotor can be expressed in Hela cells. They had different luciferaseactivities, and the expression of pGL3-GHS was significantly higher than ofpGL3-N.Conclusion These findings suggest that the single nucleotide polymorphismin 5'-regulation region of the VDR gene may play a significant role inmechanism of Idiopathic hypercalciuria. Objective To clone the promoter of VDR gene and construct luciferase reportergene vectors containing different VDR promoter fragments, and to analyze thepromoter activity of various constructions using Hela cells.Methods PCR, construct luciferase reporter gene vectors, transienttransfection and luciferase assay were used.Results Three different fragments of VDR promoter of rat were gotten. Thenthey were cloned into pGL3-Basic luciferase expression vector. These threeVDR promoter luciferase reporter gene vectors had different luciferaseactivity after transfection into Hela cells respectively.Conclusion Rat VDR promoter luciferase reporter gene plasmids weresuccessfully constructed, which will provide a useful tool for study of themolecular mechanism of VDR expression in future.