OBJECTIVE: To identify the miRNAs targeting vitamin D receptor (VDR) gene and their effect on parathyroid hormone (PTH) secretion in secondary hyperparathyroidism. METHODS: Primary parathyroid cells with secondary hyperparathyroidism were isolated by collagenase digestion and cultured. The miRNAs targeting VDR were screened by bioinformatics methods and full transcriptome sequencing, and dual-luciferase reporter assay was used to verify the targeting relationship between VDR and the screened miRNA. The effects of overexpression or inhibition of the candidate miRNA on VDR mRNA and protein expressions and PTH secretion were evaluated using qRT-PCR and Western blotting. The expression levels of the candidate miRNAs and VDR mRNA in clinical specimens of parathyroid tissues were verified by qRT-PCR, and the expression of VDR protein was detected by immunohistochemistry. RESULTS: We successfully isolated primary parathyroid cells. Dual-luciferase reporter assay verified the targeting relationship of hsa-miR-149-5p, hsa-miR-221-5p, hsa-miR-222-3p, hsa-miR-29a-5p, hsa-miR-301a-5p, hsa-miR-873-5p, hsa-miR-93-3p with VDR, and among them, the overexpression of hsa-miR-149-5p and hsa-miR-301a-5p significantly increased PTH secretion in the parathyroid cells. In patients with secondary hyperparathyroidism, hsa-miR-149-5p was highly expressed in the parathyroid tissues (P=0.046), where the expressions of VDR mRNA (P=0.0267) and protein were both decreased. CONCLUSION The two miRNAs, hsa-miR-149-5p and hsa-miR-301a-5p, may promote the secretion of PTH in patients with secondary hyperparathyroidism by down-regulating the expression of VDR gene.
Humans ; Hyperparathyroidism, Secondary/genetics* ; MicroRNAs/metabolism* ; Parathyroid Hormone ; RNA, Messenger ; Receptors, Calcitriol/genetics*

OBJECTIVETo evaluate the effect of 25-hydroxyvitamin D(3) on the expression and distribution of vitamin D receptor in normal human bronchial epithelial cells.

METHODSMTT assay was used to assess the viability of human airway epithelial cell line 16HBE following a 24-h exposure to different concentrations of 25-hydroxyvitamin D(3). Real-time quantitative PCR, Western blotting, and immunofluorescence assay were used to observe the expression and distribution of vitamin D receptor in the cells following the exposure.

RESULTSCompared with the control cells, 16HBE cells exposed to different concentrations of 25-hydroxyvitamin D(3) exhibited no significantly increase in the expression or distribution of vitamin D receptor.

CONCLUSIONThe influence of 25-hydroxyvitamin D(3) on bronchial epithelial cells might be independent of the expression and translocation of vitamin D receptor.

Bronchi ; cytology ; Calcifediol ; pharmacology ; Cell Line ; Epithelial Cells ; cytology ; metabolism ; Humans ; RNA, Messenger ; genetics ; metabolism ; Receptors, Calcitriol ; genetics ; metabolism

Reports describing significant health risks due to inadequate vitamin D status continue to generate considerable interest amongst the medical and lay communities alike. Recent research on the various molecular activities of the vitamin D system, including the nuclear vitamin D receptor and other receptors for 1,25-dihydroxyvitamin D and vitamin D metabolism, provides evidence that the vitamin D system carries out biological activities across a wide range of tissues similar to other nuclear receptor hormones. This knowledge provides physiological plausibility of the various health benefits claimed to be provided by vitamin D and supports the proposals for conducting clinical trials. The vitamin D system plays critical roles in the maintenance of plasma calcium and phosphate and bone mineral homeostasis. Recent evidence confirms that plasma calcium homeostasis is the critical factor modulating vitamin D activity. Vitamin D activities in the skeleton include stimulation or inhibition of bone resorption and inhibition or stimulation of bone formation. The three major bone cell types, which are osteoblasts, osteocytes and osteoclasts, can all respond to vitamin D via the classical nuclear vitamin D receptor and metabolize 25-hydroxyvitamin D to 1,25-dihydroxyvitamin D to activate the vitamin D receptor and modulate gene expression. Dietary calcium intake interacts with vitamin D metabolism at both the renal and bone tissue levels to direct either a catabolic action on the bone through the endocrine system when calcium intake is inadequate or an anabolic action through a bone autocrine or paracrine system when calcium intake is sufficient.
Calcium/metabolism ; Fractures, Bone/metabolism/pathology ; Humans ; Osteoporosis/metabolism/pathology ; Protein Binding ; Receptors, Calcitriol/genetics/metabolism ; Vitamin D/analogs & derivatives/*metabolism

OBJECTIVETo determine the relationship between vitamin D receptor (VDR) gene polymorphism and osteoporosis in postmenopausal women.

METHODSThe polymerase chain reaction-restriction fragment length polymorphism was used to detect VDR genotype in 40 patients with osteoporosis and 21 healthy postmenopausal women.

RESULTSIn the patients with osteoporosis, the bb, Bb, and BB genotype accounted for 82.5%, 17.5% and 0, respectively; in healthy groups, they were 85.71%, 14.29% and 0, respectively (P>0.05).

CONCLUSIONSignificant association between VDR genotype and osteoporosis in Chinese women was observed in this study.

DNA ; genetics ; metabolism ; Deoxyribonucleases, Type II Site-Specific ; metabolism ; Female ; Gene Frequency ; Genotype ; Humans ; Middle Aged ; Osteoporosis, Postmenopausal ; genetics ; Polymorphism, Genetic ; Receptors, Calcitriol ; genetics

Animals ; Bone Density ; Bone and Bones ; metabolism ; Calcium ; metabolism ; Genetic Predisposition to Disease ; Humans ; Osteoporosis ; genetics ; Polymorphism, Genetic ; Receptors, Calcitriol ; genetics ; Rickets ; etiology ; genetics

OBJECTIVE: There is asingle nucleotide polymorphism (SNP) in the exon 2 of the vitamin D receptor (VDR) gene that can be distinguished using the restriction endonuclease FokI, and accordingly divided into three genotypes: FF, Ff and ff. VDR-FokI polymorphism was the only known SNP that could alter the protein structure of VDR. CYP24A1 is the gene encoding vitamin D 24 hydroxylase and is a vitamin D responsive gene. The influence of rs2228570 on transcriptional activation by VDR in human gingival fibroblasts (hGF) and periodontal ligament cells (hPDLC) was investigated in this study. METHODS: hGF and hPDLC of 12 donors' were primarily cultured and genomic DNA was extracted. A part of genomic DNA with the length of 267 bp was obtained using PCR, which contained the SNP. VDR-Fok I genotypes were determined according to the results of restriction fragment length polymorphism. hGF and hPDLC were stimulated with 10 nmol/L 1α,25 dihydroxy vitamin D₃ (1,25OH₂D₃) or 1 000 nmol/L 25 hydroxy vitamin D₃ (25OHD₃) for 48 h before RNA was extracted. Then VDR antagonist ZK159222 was used or not used during 1,25OH₂D₃ or 25OHD₃ stimulation with hGF and hPDLC. After 1,25OH₂D₃ stimulation for 48 h, the proteins in hGF and hPDLC were also collected. The protein expressions of CYP24A1 and VDR were detected using Western blot. RESULTS: Among the 12 donors' cell cultures, the number of FF, ff and Ff genotypes was 4, 3 and 5, respectively.After stimulation with 1,25OH₂D₃ or 25OHD₃ for 48 h,CYP24A1 mRNA levels in FF-hGF were significantly higher than those in other hGF genotypes(1,25OH₂D₃: F=31.147, P<0.01; 25OHD₃: F= 32.061,P <0.01), as was in FF-hPDLC (1,25OH₂D₃: F=23.347, P<0.01; 25OHD₃: F=32.569,P<0.01). When ZK159222 was used before 1,25OH₂D₃ stimulation, this statistically significant difference disappeared (hGF: F=0.246, P=0.787; hPDLC: F=0.574, P=0.583). When ZK159222 was used before 25OHD₃ stimulation, the trend was similar (hGF: F=1.636, P=0.248; hPDLC: F=0.582, P=0.578).After stimulation with 1,25OH₂D₃ for 48 h, CYP24A1 protein levels in FF-hGF were significantly higher than those in the other hGF genotypes (F=12.368, P <0.01), as was in FF-hPDLC (F=15.749, P <0.01). In hGF and hPDLC, the mRNA or protein expression of VDR of different genotypes was not significantly different under different stimulation conditions.The paired comparison showed that there was no statistically significant difference between the expression of CYP24A1 in hGF and that in hPDLC under all the stimulation conditions, as was the expression of VDR. CONCLUSION In hGF and hPDLC, the FF-VDR genotype is associated with the more remarkable up-regulation of CYP24A1than the other genotypes, indicating that transcriptional activation of FF-VDR might be higher than those of other vitamin D receptors.
Fibroblasts/metabolism* ; Genotype ; Humans ; Periodontal Ligament/metabolism* ; Polymorphism, Genetic ; Polymorphism, Restriction Fragment Length ; Polymorphism, Single Nucleotide ; Receptors, Calcitriol/genetics* ; Vitamin D3 24-Hydroxylase/metabolism*

The genetic alterations of vitamin D receptor (VDR) are related with the growth of long bone. There were a lot of reports regarding an association of polymorphisms in the VDR promoter with many disorders, but not with idiopathic short stature (ISS). We investigated the association of them with ISS. A total of 50 subjects, including 29 ISS patients and 21 healthy controls with their heights within the normal range was recruited. We selected two single nucleotide polymorphisms (SNPs) from VDR promoter (rs11568820 at the Cdx-2 binding site upstream of exon 1e and rs4516035 at -1012 upstream of exon 1a) as candidates, respectively. In genotype analysis, the frequency of A/A genotype at the Cdx-2 binding site locus (rs11568820) upstream of exon 1e of VDR was decreased to 6.9% in ISS patients (28.6% in controls) (P = 0.027). The genetic variation at the Cdx-2 binding site of VDR promoter can be a contributing factor of growth of height.
Adolescent ; Alleles ; Binding Sites ; Child ; Dwarfism/*genetics ; Exons ; Female ; Gene Frequency ; Genotype ; Homeodomain Proteins/metabolism ; Humans ; Male ; *Polymorphism, Single Nucleotide ; Promoter Regions, Genetic ; Receptors, Calcitriol/*genetics

OBJECTIVETo investigate the prevalence of vitamin D receptor (VDR) gene polymorphism in pre-menopausal women in Guangzhou and study its relationship with bone mineral density(BMD).

METHODSThe genotypes of VDR gene in 193 per-menopausal women in Guangzhou were analyzed by polymerase chain reaction-restriction fragment length polymorphism. BMD of the lumbar spine, femoral neck, greater trochanter and Ward's triangle were measured by dual-energy X-ray absorptiometry.

RESULTSIn the 193 subjects, 120 (66.2%) were identified as VDR bb genotype, 64 (33.2%) as Bb, and 9 (4.6%) as BB. The b allele frequencies reached 78.76%, and B allele frequencies was 21.24%. The distribution followed the Hardy-Weinberg equilibrium. No significant difference was found in BMD among the subjects with different genotypes.

CONCLUSIONVDR genotype is not related to BMD, and VDR polymorphism can not be used as a genetic marker for predicting the risk of osteoporosis in pre-menopausal women in Guangzhou.

Absorptiometry, Photon ; Adult ; Analysis of Variance ; Bone Density ; China ; Female ; Gene Frequency ; Genotype ; Humans ; Middle Aged ; Osteoporosis ; genetics ; metabolism ; Polymorphism, Genetic ; Premenopause ; Receptors, Calcitriol ; genetics

It is well known that estrogen can inhibit bone absorption, decrease bone turnover and preserve bone mass. Some studies indicated that the effect of estrogen on calcium and bone is relative to vitamin D system, while others also reported that this effect of estrogen is independent of vitamin D. The genomic effect of 1alpha, 25(OH)(2)D(3)is mediated by the nuclear vitamin D receptor (VDR) in a ligand-dependent manner. Hypocalcemia, hyperparathyroidism and osteomalacia are developed in VDR gene knockout mice. To determine whether the effect of estrogen on calcium and bone is dependent on VDR, this study examined the effect of exogenous estrogen on calcium and bone homeostasis in VDR gene knockout mice. Male and female wild type (WT) and VDR gene knockout heterozygous mice were mated each other and the genotyping of their offsprings were determined by PCR. At age of 21-day, WT and knockout mice were weaned and treated by one of three different regimens: (1) WT-vehicle group: the WT mice were injected with normal saline; (2) VDR KO-vehicle group: the VDR gene knockout mice were injected with normal saline; (3) VDR KO-E group: the VDR gene knockout mice were subcutaneously injected with estradiol, 0.2 mug per mouse, once daily for 1 month. The bone mineral density (BMD) of mice was measured using dual-energy X-ray absorptiometry. All mice were sacrificed at age of 50-day. Blood was taken by heart puncture under anesthesia and serum calcium was measured by autoanalyser.Tibiae were removed, fixed and embedded with the methylmethacrylate (MMA), and undecalcified sections were cut. These sections were stained for mineral with the von Kossa staining procedure and counterstained with toluidine blue. Static histomorphometric analyses were performed on those stained sections. The results showed that the serum calcium level was (2.10+/-0.37) mmol/L in the VDR KO-vehicle mice and rose to (2.80+/-0.41) mmol/L in the VDR KO-E mice although it was still lower than WT-vehicle mice [(3.10+/-0.48) mmol/L]. BMD and mineralized trabeculer volume were increased significantly in VDR KO-E group compared with that in VDR KO-vehicle group. These results suggest that exogenous estrogen can improve calcium absorption and skeletal mineralization in a VDR-independent manner.
Animals ; Bone Density ; Calcification, Physiologic ; drug effects ; Calcium ; metabolism ; Estrogens ; pharmacology ; Female ; Gene Knockout Techniques ; Homeostasis ; Mice ; Mice, Knockout ; Receptors, Calcitriol ; genetics

Objective: To investigate the effect of small interfering RNA silencing the vitamin D receptor (VDR) on the biological behavior of prostate cancer PC-3 cells. METHODS: We constructed the VDR-shRNA lentiviral vector and determined the mRNA and protein expressions of VDR by RT-PCR and Western blot. Using scratch wound healing and Transwell chamber assays, we detected the changes in the migration and invasiveness of the PC-3 cells after silencing VDR. RESULTS: The VDR-shRNA plasmid significantly interfered the VDR expression and successfully screened the cell lines with stable VDR-shRNA interference. The rate of scratch wound healing was markedly lower in the VDR interference group than in the blank control and LV3 negative control groups (59% vs 73.6% and 77.8%, P <0.05), but with no statistically significant difference between the latter two (P >0.05), and so was the count of permeable cells (P <0.05), but with no significant difference between the latter two groups, either (P >0.05). The migration ability and invasiveness of the VDR-treated cells were remarkably decreased as compared with those of the control cells. CONCLUSIONS Down-regulated expression of the VDR gene may reduce the migration and invasiveness of prostate cancer cells.
Cell Line, Tumor ; Cell Movement ; genetics ; Cell Proliferation ; Down-Regulation ; Gene Silencing ; Humans ; Lentivirus ; Male ; Neoplasm Invasiveness ; genetics ; Plasmids ; Prostatic Neoplasms ; genetics ; pathology ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; Receptors, Calcitriol ; genetics ; metabolism ; Transfection ; Wound Healing ; genetics