Vitamin D Receptor mRNA Research Articles

Molecular investigation of vitamin D receptor (VDR) genetic variants and their impact on VDR mRNA and serum vitamin D levels in allergic rhinitis in an Indian population: A case-control study.

Vitamin D deficiency is widespread and poses a significant health concern, as emerging research links it to allergic diseases owing to its immunomodulatory functions. The optimal functioning of vitamin D and its activation depend on its nuclear receptor, vitamin D receptor (VDR). Genetic variants of VDR have been explored as potential factors in autoimmune and allergic diseases, with limited studies on their association with allergic rhinitis (AR). The present investigation aims to analyse the role of three VDR genetic variants - TaqI, FokI and BsmI - in AR susceptibility and their impact on VDR mRNA and serum vitamin D levels. A total of 550 subjects, consisting of 250 AR cases and 300 age- and gender-matched controls, underwent genotyping by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). VDR mRNA and vitamin D levels were determined by quantitative real-time PCR and chemiluminescence, respectively. Although TaqI did not exhibit significant differences, FokI demonstrated a noteworthy association with AR, particularly with the CC genotype (odds ratio [OR]: 3.34; confidence interval [CI]: 1.79-6.23). Similarly, BsmI revealed an increased risk for AR, with the GA+AA genotypes showing a 2.2-fold elevated risk (OR: 2.20; CI: 1.53-3.16). VDR mRNA expression was threefold lower in AR patients (p<.0001), accompanied by reduced serum vitamin D levels (p<.0001). In addition, CC (p=.01) and AA (p=.02) genotypes of FokI and BsmI were associated with reduced VDR mRNA levels, whereas TaqI showed no such association. Similarly, heterozygous genotypes of TaqI and FokI, as well as homozygous AA of BsmI, correlated with lower serum vitamin D levels (p<.001). This study emphasizes the intricate relationship among VDR genetic variations, altered VDR activity, immune modulation and vitamin D metabolism in AR. Further research involving diverse populations is crucial for confirming and generalizing these findings, paving the way for personalized therapeutic interventions in vitamin D-related disorders.

VDR mRNA Expression And Serum Vitamin D Levels in Post-Covid Vaccinated Patients

The emergence of COVID-19 vaccines has reshaped the trajectory of the ongoing pandemic, offering hope for widespread immunity. Beyond conferring protection against SARS-CoV-2, these vaccines have exhibited intriguing immunomodulatory effects. This research explores the dynamic interplay among VDR mRNA expression levels, calcium (ionized and total), and intact parathyroid hormone (iPTH) concentrations in individuals post-COVID-19 vaccination. The Vitamin D Receptor (VDR) plays a pivotal role in immune regulation and is closely intertwined with calcium homeostasis. This study investigates the hypothesis that COVID-19 vaccination may induce alterations in VDR mRNA expression, subsequently influencing calcium metabolism and iPTH secretion. Our findings reveal dynamic shifts in VDR mRNA expression following COVID-19 vaccination, with distinct patterns observed across individuals. Concurrently, we observe ionized and total calcium levels alterations, hinting at potential links between VDR activity and calcium metabolism post-vaccination. Furthermore, iPTH levels exhibit intriguing fluctuations, suggesting a regulatory role of VDR in parathyroid hormone secretion. The integration of clinical outcomes and vaccine response data sheds light on the significance of these molecular and biochemical alterations. This research underscores the multifaceted impact of COVID-19 vaccination on VDR mRNA expression, calcium homeostasis, and iPTH regulation. Beyond the scope of vaccination, our findings may bear implications for immunomodulation in various disease contexts, particularly in individuals with pre-existing calcium-related disorders. In conclusion, our study unveils the intricate relationships among VDR mRNA expression, calcium levels, and iPTH concentrations in the context of post-COVID-19 vaccination. These discoveries extend our understanding of vaccine-induced immunomodulation and may pave the way for personalized vaccination strategies, while also opening new avenues for investigating the role of VDR in immune responses and calcium regulation beyond the pandemic.

Gene expression profiling of vitamin D metabolism enzymes in leukemia and lymphoma patients: molecular aspect interplay of VDR, CYP2R1, and CYP24A1.

Vitamin D deficiency is prevalent among the Indonesian population, particularly in individuals diagnosed with leukemia-lymphoma. The regulation of vitamin D metabolism is influenced by the expression of several enzymes, such as CYP2R1, CYP24A1, and the vitamin D receptor (VDR). This study aimed to scrutinize the gene expression profiles in both mRNA and protein levels of VDR, CYP2R1, and CYP24A1 in leukemia and lymphoma patients. The research was a cross-sectional study conducted at Cipto Mangunkusumo Hospital (RSCM) in Jakarta, Indonesia. The study included a total of 45 patients aged over 18years old who have received a diagnosis of lymphoma or leukemia. Vitamin D status was measured by examining serum 25 (OH) D levels. The analysis of VDR, CYP2R1, and CYP24A1 mRNA expression utilized the qRT-PCR method, while protein levels were measured through the ELISA method. The study revealed a noteworthy difference in VDR protein levels between men and women. The highest mean CYP24A1 protein levels were observed in the age group > 60years. This study found a significant, moderately positive correlation between VDR protein levels and CYP24A1 protein levels in the male and vitamin D sufficiency groups. In addition, a significant positive correlation was found between VDR mRNA levels and CYP2R1 mRNA levels, VDR mRNA levels and CYP2R1 mRNA levels, and CYP2R1 mRNA levels and CYP24A1 mRNA levels. However, the expression of these genes does not correlate with the protein levels of its mRNA translation products in blood circulation.

Vitamin D status alters genes involved in ovarian steroidogenesis in muskrat granulosa cells

This study aims to explore the relationship between altered vitamin D (VitD3) status and ovarian steroidogenesis in muskrats during the breeding and non-breeding seasons. During the breeding season, the ovaries of muskrats were observably enlarged and increased in weight, accompanied by elevated serum and ovarian VitD3 status. Vitamin D receptor (VDR), VitD3 metabolic molecules (CYP2R1, CYP27B1, and CYP24A1), and steroidogenic enzymes were immunolocalized in the ovarian cells of muskrats. The mRNA levels of VDR, CYP2R1, CYP27B1, and steroidogenic enzymes were considerably higher during the breeding season compared to the non-breeding season. RNA-seq analysis revealed a prominent enrichment of vitamin-related and ovarian steroidogenesis pathways. Furthermore, the addition of 1,25(OH)2D3 to the muskrat granulosa cells in vitro increased VDR and steroidogenic enzymes mRNA levels and enhanced the 17β-estradiol level. Overall, these findings supported that VitD3 promotes the secretion of steroid hormones, thereby affecting seasonal changes in ovarian function in the muskrats.

Exercise-induced vitamin D receptor and androgen receptor mediate inhibition of IL-6 and STAT3 in muscle

BackgroundSkeletal muscle produces interleukin-6 (IL-6) during exercise as a myokine. Although IL-6 is required for skeletal muscle regeneration, its action increases the expression of myostatin and other proteins involved in muscle atrophy, resulting in skeletal muscle atrophy. In this study, we clarified the effects exercise-induced vitamin D receptor (VDR) and androgen receptor (AR) expression on IL-6 and signal transducer and activator of transcription 3 (STAT3) in vivo and in vitro. MethodC2C12 myotubes were subjected to electric pulse stimulation (EPS) in vitro. To evaluate VDR and AR function, a VDR/AR agonist and antagonist were administered before EPS to C2C12 myotubes. C57BL6 mice underwent 4 weeks of exercise. The expression levels of proteolytic-associated genes, including CCAAT/enhancer-binding protein delta (C/EBPδ) and myostatin, were measured by quantitative real-time polymerase chain reaction, and phosphorylated and total STAT3 levels were measured by Western blot analysis. ResultThe expression of VDR and AR mRNA was induced following EPS in C2C12 myotubes. IL-6 mRNA expression was also increased with a peak at 6 h after EPS and p-STAT3/STAT3 ratio reciprocally decreased. Although VDR/AR agonist administration decreased IL-6 mRNA expression and p-STAT3/STAT3 ratio, these two endpoints increased after treatment with VDR/AR antagonist, respectively. Exercise in mice also increased the expression of VDR/AR and IL-6 mRNA and decreased p-STAT3/STAT3 ratio. ConclusionExercise-induced VDR and AR expression results in the suppression of IL-6 mRNA and STAT3 phosphorylation in skeletal muscle.

High Vitamin D Level in Female Mice Increases the Number of Live Fetuses.

Little is known about the impact of high-normal range of 25-hydroxyvitamin D [25(OH)D] on reproductive function. The aim of this study was to investigate the effect of different dose vitamin D supplementation in female mice on the pregnancy outcomes. Three groups of female mice were fed with fodder containing different dose of vitamin D at both pre-gestational and gestational stages. Serum 25(OH)D and calcium concentrations were monitored. The expression levels of vitamin D receptor (VDR) mRNA and protein in placenta were determined by real-time RT-PCR and western blot. Pregnancy outcomes were evaluated and compared among the three groups. Compared with the medium and low dose groups, serum 25(OH)D concentration was significantly increased and approximated to high-normal range in the high dose group (pre-gestational: 81.3±5.75 vs 52.8±6.24 and 25.0±3.99 ng/mL; gestational: 86.8±5.99 vs 52.6±9.29 and 27.9±4.96 ng/mL, respectively; all p<0.001). Interestingly, the average number of live fetuses per litter was much larger in the high dose group than in other two groups (19.8±5.31 vs 13.8±1.30 and 12.8±3.55 respectively, both p<0.05). However, no significant differences of the expression levels of VDR mRNA and protein in placenta were identified among the three groups. Supplementation of high dose vitamin D can enhance the female mice reproductive function. Further study is warranted to explore the mechanism by which high level of 25(OH)D in female mice increases the number of fetuses.

MicroRNA-125b mediates Interferon-γ-induced downregulation of the vitaminD receptor in systemic lupus erythematosus.

The inflammatory factor interferon (IFN)-γ is related to the occurrence and development of systemic lupus erythematosus (SLE). The vitaminD receptor (VDR) has an anti-inflammatory effect and its downregulation is involved in the onset of SLE. Our previous studies have confirmed that the expression of VDR in SLE peripheral blood mononuclear cells (PBMCs) is downregulated, which is negatively correlated with disease activity and inflammation. However, the mechanism underlying VDR downregulation in SLE is unknown. Based on the results of computer simulation analysis, the expression of VDR and four microRNAs (miR-17-3p, miR-34a, miR-346, and miR-125b) in SLE PBMC cells was analyzed under proinflammatory cytokine IFN‑γ treatment, and miR-125b was identified as the target miRNA. The relationship between IFN‑γ, miR-125b, and VDR was further assessed in THP‑1 cells. We showed that IFN‑γ inhibited the expression of VDR and miR-125b. Further study revealed that VDR mRNA was positively correlated with miR-125b in THP‑1 cells after IFN‑γ intervention. After transfection of miR-125b mimic or inhibitor, the expression of VDR in the miR-125b inhibitor group was lower than in the control group and miR-125b mimic group, while expression in the control group was lower than in miR-125b mimic group. Transfection of miR-125b inhibitor into THP‑1 cells could further promote the ability of IFN‑γ to inhibit VDR. The decrease in VDR expression promotes development of inflammation and SLE. These data suggest that miR-125b may mediate inflammatory factor IFN-γ-induced downregulation of VDR in the pathogenesis of SLE.

Clinical efficacy of moxibustion for ulcerative colitis and its influence on vitamin D receptor

ObjectiveTo observe the clinical efficacy of herbal cake-partitioned moxibustion for ulcerative colitis (UC) and elucidate its mechanism by targeting the vitamin D receptor (VDR) signaling pathway.MethodsA total of 63 patients with UC were randomly divided into an observation group (30 cases, treated with herbal cake-partitioned moxibustion) and a control group (33 cases, treated with sham herbal cake-partitioned moxibustion). Moxibustion treatment was performed at Qihai (CV6) and bilateral Tianshu (ST25) and Shangjuxu (ST37), 3 times per week for 12 weeks. The total effective rate, visual analog scale (VAS) score for abdominal bloating and pain, and hospital anxiety and depression scale (HADS) score were compared between the two groups. Enzyme-linked immunosorbent assay was used to detect the concentrations of serum C-reactive protein (CRP), 25-hydroxyvitamin D [25(OH)D], and interleukin-12 (IL-12)/interleukin-23 (IL-23) p40. Immunohistochemistry was used to observe the expression levels of VDR and regenerating gene IV (Reg IV) proteins in colonic mucosa. The expression levels of VDR, cytochrome p450 27B1 (CYP27B1), and Reg IV mRNAs were detected by real-time fluorescence quantitive polymerase chain reaction.ResultsAfter treatment, the total effective rate in the observation group was 86.7%, which was significantly higher than 51.5% in the control group (P<0.05). After treatment, the VAS scores for abdominal bloating and pain in the observation group were significantly decreased (P<0.01), as well as the HADS-depression subscale (HADS-D) and HADS-anxiety subscale (HADS) scores (P<0.05), while only the VAS score for abdominal pain in the control group was reduced (P<0.05), and the improvements of the scores in the observation group were more significant than those in the control group (P<0.05). After treatment, the serum CRP concentrations in both groups and the IL-12/IL-23 p40 concentration in the observation group were significantly decreased (P<0.05), and the concentrations in the observation group were lower than those in the control group (P<0.05). The expression levels of VDR protein and mRNA in the colon in both groups were all increased (P<0.01), and the expression levels of Reg IV protein and mRNA and CYP27B1 mRNA were all decreased in the two groups (P<0.05 or P<0.01); the improvements in the observation group were more notable than those in the control group (P<0.05 or P<0.01).ConclusionHerbal cake-partitioned moxibustion can effectively alleviate abdominal pain and diarrhea in patients with UC, improve depression and anxiety disorders, and regulate the expression of related proteins in the VDR signaling pathway. The mechanism may be related to inhibiting intestinal inflammation by reducing the release of the proinflammatory cytokine IL-12/IL-23 p40.

Aerobic Exercise Ameliorates Myocardial Fibrosis via Affecting Vitamin D Receptor and Transforming Growth Factor-β1 Signaling in Vitamin D-Deficient Mice.

Myocardial fibrosis is a pathological phenomenon associated with cardiovascular disease (CVD) that plays a crucial role in the development of heart diseases. Vitamin D deficiency can promote the development of CVD and exercise plays a role in the treatment of CVD. This study aimed to explore the effects of 12-week aerobic exercise training on myocardial fibrosis in vitamin D-deficient mice. A vitamin D-deficient mouse model was induced by a vitamin D-deficient (0 IU Vitamin D3/kg) diet. Twenty-four C57BL/6J male mice were randomly divided into three groups: a control sedentary group (CONS, n = 8), a vitamin D-deficient sedentary group (VDDS, n = 8), and a vitamin D-deficient exercise group (VDDE, n = 8) which was aerobically trained for 12 weeks. The results showed that the serum 25-hydroxyvitamin D [25(OH)D] levels of the VDDS group were <50 nmol/L, which was significantly lower than that of the CONS group. Compared with the CONS group, the VDDS group showed cardiac dysfunction and significant fibrosis, together with lower vitamin D receptor (VDR) mRNA and protein expression levels, higher mRNA expression levels of profibrotic and inflammatory factors, and higher transforming growth factor-β1 (TGF-β1) and phospho-Smad2/3 (P-Smad2/3) protein expression levels. Serum 25(OH)D levels in the VDDE group were significantly higher than those in the VDDS group. Compared with the VDDS group, the VDDE group showed improved cardiac function and alleviated myocardial fibrosis. Meanwhile, the VDDE group had significantly higher VDR mRNA and protein expression levels; lower mRNA expression levels of profibrotic and inflammatory factors; and lower TGF-β1 and P-Smad2/3 protein expression levels. In conclusion, aerobic exercise training remains a promising intervention for treating myocardial fibrosis in vitamin D deficiency.

Vitamin D receptor expression in sperm of males with unexplained infertility.

Vitamin D is associated with numerous disorders, including infertility. Accordingly, the goal of this research was to find out the level of vitamin D and messenger RNA (mRNA) expression of vitamin D receptor (VDR) in the sperm of male subjects with unexplained infertility. Twenty-four unexplained infertile men as the case group and 22 healthy fertile men as the control group were recruited. Vitamin D levels were evaluated in seminal fluid using enzyme-linked immunosorbent assay (ELISA). Afterwards, the swim-up test was performed to isolate motile sperm cells. From these cells, RNA was extracted, complementary DNA (cDNA) was synthesized, and mRNA expression of the VDR gene was evaluated with quantitative real-time polymerase chain reaction (PCR). A decrease in VDR mRNA expression levels was detected in the case group compared to the control group, but this reduction was not statistically significant (p>0.05). Besides, the level of vitamin D in seminal fluid was not detectable in both groups. The sperm of unexplained infertile men express VDR gene mRNA, although there was no vitamin D in seminal samples. Hence, vitamin D and VDR signaling might not be effective in the etiopathogenesis of unexplained infertility in men.

Vitamin D Receptor (VDR) Genetic Variants: Relationship of FokI Genotypes with VDR Expression and Clinical Disease Activity in Systemic Lupus Erythematosus Patients.

Vitamin D (VD) deficiency is more frequent in systemic lupus erythematosus (SLE) patients than in control subjects (CS); genetic variants in the VD receptor (VDR) could contribute to the clinical disease activity. This study was aimed to determine the association of the VDR variants FokI (rs2228570), BsmI (rs1544410), ApaI (rs7975232), and TaqI (rs731236) with susceptibility to the disease, VD status, VDR mRNA expression, and clinical disease activity in SLE patients. A cross-sectional study was conducted in 194 SLE and 196 CS Mexican women. Immunoassays quantified serum calcidiol and calcitriol. Genotyping was performed by allelic discrimination assays and mRNA VDR expression by qPCR. The FokI variant was not in linkage disequilibrium with BsmI, ApaI, and TaqI&nbsp;VDR variants. SLE patient carriers of the TT FokI genotype showed higher clinical disease activity scores. Notably, the mRNA VDR expression was higher in SLE patients vs. CS, in active vs. inactive SLE patients, and in participants of both study groups with vitamin D deficiency, higher calcitriol levels, and TT FokI genotype carriers. In conclusion, the TT FokI VDR genotype was related to high VDR expression and clinical disease activity in systemic lupus erythematosus patients.

OR21-4 Comparison of the Effects of PTH (1-34), PTHrP (1-36) and Abaloparatide (ABL) on the Murine Osteoblast Transcriptome

Abstract Osteoporosis is a prevalent disease with substantial morbidity/mortality among the aging population. Due to gaps in knowledge, current therapeutics are limited in their ability to prevent degeneration of bone while also stimulating formation of new bone. Teriparatide (PTH (1-34)) and its analogs PTHrP (1-36) and abaloparatide (ABL), have been utilized for treatment of osteoporosis but have significant limitations in efficacy over long-term use. Research from our laboratory has shown PTH (1-34), PTHrP (1-36), and ABL exert time and dose-dependent differential responses in the osteoblast, leading us to hypothesize they may also differentially modulate the osteoblast transcriptome. In this study we show that treatment of mouse calvarial osteoblasts with 1 nM of these peptides for 4 h results in differing effects on the osteoblast transcriptome by performing gene enrichment analysis of RNA-Seq data. Genes were selected with a Log2 fold change &amp;gt;1 and a false discovery rate &amp;lt;0.05. These data were analyzed and compiled into heat maps for each peptide and smear/volcano plots. RNA-Sequencing revealed that PTH (1-34) regulated 367 genes, 194 were unique; PTHrP (1-36) regulated 117 genes, 15 were unique; ABL regulated 179 genes, 20 were unique. There were 74 genes shared only among PTH(1-34) and ABL; 16 genes shared only among PTH (1-34) and PTHrP; and 83 genes shared among all three peptides. Data collected show pathway-specific differences including, 1) cAMP/PKA, 2) Wnt/β-catenin, 3) Transcriptional regulation 4) Inflammatory response 5) Transmembrane transport, 6) Metabolism, and 7) NF-kB. Further analysis of the data illuminated that the three peptides increased Vitamin D receptor (VDR) and Cbp/p300-interacting transactivator 1 (CITED1) mRNAs similar to RankL expression. These findings were confirmed via qRT-PCR of additional cultured samples of mouse calvarial osteoblasts, treated with 1 nM of PTH (1-34), PTHrP (1-36), and ABL for 4 h prior to harvest. RankL mRNA, VDR mRNA, and CITED1 mRNA were measured for each sample and statistical differences were analyzed via Kruskal Wallis p&amp;lt;0.05. Additionally, we analyzed mRNA levels of several genes of interest, including WNT7b, WNT11, TCF7, SFRP4, FZD5, PP2R2A, and DVL3 mRNA. Pathway analysis and subsequent qRT-PCR confirmation has shown that PTH (1-34) and ABL lead to a significant increase in Wnt11 mRNA, while PTHrP (1-36) does not. Our findings highlight the complexity of the genetic and functional events triggered by PTH (1-34) and its analogs. Many studies have examined PTH signaling in the osteoblast/osteocyte; ours is the first to examine global effects of these peptides on the osteoblast transcriptome. Further delineation of which signaling events are attributable to PTH (1-34), PTHrP (1-36), and ABL exclusively and which are shared among all three will further our understanding of the effects these peptides have on the osteoblast and lead to refinement of PTH-derived treatments for osteoporosis. Presentation: Monday, June 13, 2022 11:45 a.m. - 12:00 p.m.

Hepatic vitamin D receptor expression is negatively associated with liver inflammation and fibrosis in patients with chronic HBV infection.

The vitamin D receptor (VDR) is a nuclear transcription factor that acts as the main transducer in response to vitamin D (VD), regulating about 3% of thegene expression inthehumangenome. This study investigated the expression of VDR in the liver of patients with chronic hepatitis B virus (HBV) infection and determined its correlation with liver inflammation and fibrosis. We evaluated the effects of HBV infection on the expression of VDR in vivo and in vitro and further investigate the potential mechanism. Subsequently, the associations between hepatic VDR expression with liver inflammation and fibrosis were statistically analyzed. Results showed that hepatic VDR expression was significantly decreased in patients with chronic HBV infection as compared to healthy individuals. Similarly, in vitro experiments further confirmed that HBV infection could inhibit the expression of VDR in hepatocytes. Mechanistically, HBV was able to directly induce the expression of miR-125a which inhibited the mRNA and protein levels of VDR. Statistical analysis showed that hepatic VDR expression was significantly negatively correlated with liver inflammation and fibrosis in patients. We conclude that inhibition of hepatic vitamin D receptor expression by HBV/miR-125a is negatively associated with liver inflammation and fibrosis in patients with chronic HBV infection.

MiR-27a-3p and miR-30b-5p inhibited-vitamin D receptor involved in the progression of tuberculosis.

BackgroundMicroRNAs (miRNAs) play a vital role in tuberculosis (TB). Vitamin D receptor (VDR), an miRNA target gene, and its ligand, vitamin D3 (VitD3), have been reported to exert protective effects against TB. However, whether miRNAs can affect the progression of TB by targeting VDR has not been reported.Materials and methodsResearch subjects were selected according to defined inclusion criteria. A clinical database of 360 samples was established, including the subjects’ demographic information, miRNA expression profiles and cellular experimental results. Two candidate miRNAs, miR-27a-3p, and miR-30b-5p, were identified by a high-throughput sequencing screen and validated by qRT–PCR assays. Univariate and multivariate statistical analyses were performed. VDR and NF-kB p65 protein levels were detected by Western blot assays. Proinflammatory cytokine expression levels were detected by enzyme-linked immunosorbent assay (ELISA). Luciferase assays and fluorescence-activated cell sorting (FACS) were further applied to elucidate the detailed mechanisms.ResultsDifferential miRNA expression profiles were obtained, and miR-27a-3p and miR-30b-5p were highly expressed in patients with TB. These results showed that the two miRNAs were able to induce M1 macrophage differentiation and inhibit M2 macrophage differentiation. Further experiments showed that the two miRNAs decreased the VDR protein level and increased proinflammatory cytokine secretion by macrophages. Mechanistically, the miRNAs targeted the 3′ untranslated region (3′UTR) of the VDR mRNA and thereby downregulated VDR protein levels by post-transcriptional regulation. Then, due to the reduction in VDR protein levels, the NF-kB inflammatory cytokine signaling pathway was activated, thus promoting the progression of TB.ConclusionOur study not only identified differentially expressed miRNAs between the TB and control groups but also revealed that miR-27a-3p and miR-30b-5p regulate proinflammatory cytokine secretion and macrophage differentiation through VDR in macrophages. Thus, these two miRNAs influence the progression of TB.

Altered vitamin D metabolism is involved in the dysregulation of γδT cell function and their crosstalk with trophoblasts in recurrent pregnancy loss.

Recurrent pregnancy loss (RPL) is a common disease characterized by immune dysfunction and vitamin D deficiency. This study aimed to investigate vitamin D metabolism and γδT cell phenotypes at the maternal-fetal interface in women with early normal pregnancy (NP) and RPL and to determine the effects of vitamin D on the functions of γδT cells and their crosstalk with trophoblasts. The levels of 25-(OH)VD3 , the expression of vitamin D metabolic enzymes in the villi, and the proportion of γδT cells in the decidua were detected in women with NP and RPL. After treatment with different concentrations of vitamin D, the mRNA expression of the vitamin D receptor (VDR), cytokines, and transcription factors were detected in Vδ2+ γδT cells. In addition, the proliferation, migration, and invasion of HTR-8/SVneo trophoblasts were determined by coculturing them with vitamin D-treated Vδ2+ γδT cells and their supernatants. In women with RPL, the level of 25-(OH)VD3 in the villi was increased; however, that of CYP27B1 (enzyme converting 25-(OH)VD3 into 1,25-(OH)2 VD3 ) was decreased. In addition, the proportion of Vδ2+ γδT cells increased, whereas that of Foxp3+ Vδ2+ γδT cells decreased in the decidua of women with RPL. An in vitro study showed that vitamin D increased the expression of VDR mRNA and Foxp3, but decreased the expression of IFN-γ mRNA, in Vδ2+ γδT cells. Finally, vitamin D-treated Vδ2+ γδT cells promoted trophoblast migration and invasion. Abnormal vitamin D metabolism and γδT cell proportions were present at the maternal-fetal interface in women with RPL. Under normal pregnancy conditions, vitamin D can induce the differentiation of decidual Vδ2+ γδT cells toward an anti-inflammatory phenotype (Treg-like γδT cells) and modulate the crosstalk between Vδ2+ γδT cells and trophoblasts.

Identification of microRNAs targeting vitamin D receptor and their effect on parathyroid hormone secretion in secondary hyperparathyroidism

To identify the miRNAs targeting vitamin D receptor (VDR) gene and their effect on parathyroid hormone (PTH) secretion in secondary hyperparathyroidism. 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. 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. 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.

Tanshinone IIA attenuates high glucose-induced epithelial-to-mesenchymal transition in HK-2 cells through VDR/Wnt/β-catenin signaling pathway.

The progression of diabetic kidney disease (DKD) is closely related to renal tubular epithelial- to-mesenchymal transition (EMT) and tubulointerstitial fibrosis. Tanshinone IIA (TSIIA), extracted from a traditional Chinese medicine named Salvia miltiorrhiza, has been proved to have anti-fibrosis effects. The aim of this study was to investigate the effect of TSIIA on high glucose-induced EMT in human proximal tubular cells (HK-2 cells) and its possible mechanism. The proliferation of cells exposed to different concentrations of glucose was measured by light microscopy and CCK-8 test. The cells were stimulated with 30 mM glucose and different concentrations of TSIIA (5 μM or 10 μM) for 48 h. Vitamin D receptor (VDR)-siRNA was used to transfect cells, and high glucose and TSIIA treatment were further used to treat cells. The expression of alpha smooth muscle actin (a-SMA) mRNA was detected by qPCR to ensure successful induction of EMT, and the expression of VDR mRNA was detected by qPCR to ensure successful transfection of VDR-siRNA. Protein expression of a-SMA, E-cadherin, VDR, b-catenin and glycogen synthase kinase 3b (GSK-3b) was detected by Western blot analysis. The results showed that high glucose concentration inhibited cell proliferation and promoted EMT in HK-2 cells. TSIIA could reverse high glucose-induced EMT by increasing the level of VDR protein and inhibiting the levels of b-catenin and GSK-3b proteins suggestive of a negative correlation between VDR and the Wnt/b-catenin pathway. After VDR-siRNA transfection and incubation of cells at high glucose concentration, the inhibitory effect of VDR on the expression of b-catenin and GSK-3b of Wnt pathway was suppressed and the b-catenin pathway was activated. When VDR level was restored by TSIIA, the inhibitory effect of VDR on the pathway was also restored and the activation of the pathway was suppressed. TSIIA was able to attenuate high glucose-induced EMT in HK-2 cells by up-regulating VDR levels, which might be related to the inhibitory effect of VDR on the Wnt pathway.

Placental mRNA and Protein Expression of VDR, CYP27B1 and CYP2R1 Genes Related to Vitamin D Metabolism in Preeclamptic Women

(1) Background: Considerable evidence indicates that the occurrence of preeclampsia (PE) is associated with a reduced vitamin D (VD) level. Several studies have found that VD deficiency is correlated with disturbed trophoblast invasion, reduced angiogenesis and increased vasoconstriction. Because the vitamin D receptor (VDR) and CYP27B1 and CYP2R1 hydrolases are strongly involved in VD metabolism, the goal of the present study was to evaluate their genes and proteins expression in the placentas from preeclamptic women. (2) Methods: Samples and clinical data were obtained from 100 Polish women (41 women with preeclampsia and 59 healthy pregnant controls). The whole PE group was divided into subgroups according to gestation week of pregnancy ending before and after 34 gestational weeks (early/late-onset preeclampsia (EOPE/LOPE)). However, finally, to reduce confounding by differences in gestational age, the EOPE group was excluded from the analysis of mRNA and protein placental expression, and we focus on the comparison between LOPE and control groups. The placental VDR, CYP27B1 and CYP2R1 mRNA expression was analyzed using RT-PCR, and placental protein levels were determined by ELISA assay. (3) Results. (3.1) Placental gene expression: Expression levels of both genes, CYP27B1 (1.17 vs. 1.05 in controls, p = 0.006) and CYP2R1 (2.01 vs. 1.89 in controls, p = 0.039), were significantly higher in preeclamptic placentas than in the control group. Interestingly, VDR expression was significantly lower in placentas from the PE group (1.15 vs. 1.20 in controls, p = 0.030). After dividing all preeclamptic women into subgroups only for the CYP27B1 gene, a significantly higher placental expression in the LOPE subgroup than the healthy controls was observed (padj = 0.038). (3.2) Placental protein expression: The results revealed that protein expression levels of CYP27B1 in the preeclamptic group were similar (5.32 vs. 5.23 in controls, p = 0.530). There was a significant difference in median VDR and CYP2R1 protein levels between studied groups (VDR: 2.56 vs. 3.32 in controls, p &lt; 0.001; CYP2R1: 1.32 vs. 1.43 in controls, p = 0.019). After stratification of preeclamptic women into subgroups, a significant difference was observed only in the VDR protein level. The medians in the LOPE subgroups were significantly lower compared to the healthy control group. In the whole study group, the placental VDR protein level was inversely correlated with systolic and diastolic blood pressure (all p &lt; 0.001), and positively correlated with gestational age (p &lt; 0.001) and infant birth weight (p = 0.014). (4) Conclusions: Lower mRNA and protein expression of VDR in preeclamptic placentas, and also VDR protein expression, could play a pivotal role in preeclampsia development. Additionally, the higher mRNA expression of both CYP27B1 and CYP2R1 hydrolase genes in placentas from preeclamptic women could indicate the compensatory role of these enzymes in preeclampsia etiology. Our results also indicate that placental VDR protein level could be one of the factors modulating blood pressure in pregnant women, as well as influencing gestational age and infant birth weight. Considering the importance of these findings, future studies are warranted.

Diagnostic Value of VDR in Bone Metastasis and Prognosis of Patients with Breast Cancer and Expression Correlation between Vitamin D Receptor and Hairless Protein

Background: Breast cancer is more likely to metastasize to the bone. Previous researches have revealed that the vitamin D receptor (VDR) contributes to breast cancer progression and bone metastasis in mouse and human breast cells, and hairless (Hr) protein interacts with VDR in the mammalian hair cycle. This study aimed to explore the expression of VDR/Hr in breast cancer, and the correlation between VDR/Hr and prognosis, bone metastasis, and metastasis-related prognosis. Methods: The expression of VDR and Hr was analyzed on 119 breast cancer tissues and corresponding normal breast tissue from each of the breast cancer samples by immunohistochemistry staining, and the databases were supplemented as well. Results: The expression of the VDR protein was significantly decreased in breast cancer patients (p < 0.05), inversely, the UALCAN (p = 0.000) and GEPIA (p > 0.05) databases showed that the VDR mRNA expression tended to be higher in tumor tissues. The Hr protein was expressed at a low level within breast cancer specimens (p < 0.05), which was in agreement with the level of Hr mRNA in UALCAN (p = 0.005) and GEPIA (p > 0.05). The protein levels of VDR and Hr were positively correlated (p > 0.05), while the mRNA levels suggested a close relationship with GEPIA (p < 0.05). Low expression of Hr protein displayed a tendency for longer overall survival (OS) and recurrence-free survival (RFS), and its mRNA data also revealed the same trend in the Kaplan-Meier dataset (both p > 0.05). However, VDR protein and mRNA with low expression had markedly shorter OS and RFS (both p < 0.05). The downregulation of VDR protein was significantly associated with an advanced stage (p < 0.05). Low VDR protein was an independent risk factor for poor prognosis (p < 0.05) and was negatively correlated with bone metastasis (p < 0.05). VDR protein and mRNA levels were both downregulated in breast cancer with bone metastasis (both p < 0.05). The area under ROC curve (AUC) for VDR protein expression to identify patients with bone metastasis was 0.661 (p < 0.05) and the AUC for VDR level to predict 1-year, 3-year, and 5-year OS was 0.621, 0.664, and 0.805 in patients with bone metastasis, respectively (p < 0.05). VDR with low expression accelerated bone metastasis and metastasis-related poor survival (both p < 0.05). Conclusion: VDR expression is a notable prognostic factor in primary breast cancer patients for predicting bone metastases and unfavorable clinical outcome.

The clinical significance of vitamin D levels and vitamin D receptor mRNA expression in colorectal neoplasms.

Background/AimThis study aimed to investigate the clinical significance of changes in vitamin D [25(OH)D] levels and vitamin D receptor (VDR) mRNA expression in colorectal adenoma development.MethodsPlasma concentrations of 25(OH)D and mRNA expression of VDR in tissues were determined by enzyme‐linked immunosorbent assay (ELISA) and real‐time fluorescence quantitative polymerase chain reaction (RT‐qPCR), respectively. In addition, the concentration of plasma 25(OH)D and levels of VDR mRNA in tissues were compared among healthy individuals and adenoma and adenocarcinoma patients.ResultsVitamin D receptor expression in colorectal adenocarcinoma tissues was significantly lower than that in para‐cancerous tissues that were >5 cm away from malignant tumor sites (p < 0.01). The level of VDR expression in normal colorectal tissues from healthy individuals was significantly higher than that in colorectal adenomas (p < 0.01) and colorectal adenocarcinomas (p < 0.01); however, the VDR expression was not significantly different between colorectal adenomas and colorectal adenocarcinomas (p = 0.106). The concentration of 25(OH)D in healthy individuals was significantly higher than that in patients with colorectal adenomas (p < 0.01) and colorectal adenocarcinomas (p < 0.01); however, the concentration of 25(OH)D was not significantly different between colorectal adenomas and colorectal adenocarcinomas (p = 0.489). A low concentration of 25(OH)D was considered a risk factor for colorectal adenoma and colorectal adenocarcinoma, with odds ratios of 4.875 and 2.925, respectively.ConclusionsThe 25(OH)D levels and VDR mRNA expression might be associated with the development of colorectal adenoma and its progression to adenocarcinoma.