Expression data from 1,25(OH)2D treated human testicular cells
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ABSTRACT: Human testicular cells were isolated mechanically and enzymatically from testis of braindead donors and from urological samples. The expression of genes was studied at baseline and 1,25(OH)2D treated conditions. We used microarrays to analyze the gene expression underlying vitamin D metabolism in human testis cells and identified distinct classes of up-regulated genes during this process. Testicular primary cells were treated with 100nM 1,25(OH)2D for 24h and gene expression studied by microarray on transcript level.
Project description:Human testicular cells were isolated mechanically and enzymatically from testis of braindead donors and from urological samples. The expression of genes was studied at baseline and 1,25(OH)2D treated conditions. We used microarrays to analyze the gene expression underlying vitamin D metabolism in human testis cells and identified distinct classes of up-regulated genes during this process.
Project description:Heterogeneous nuclear ribonucleoprotein C1/C2 (hnRNPC1/C2) functions as an RNA splicing regulator through co-transcriptional association with nascent mRNA. HnRNPC1/C2 can also bind to double-stranded DNA as a vitamin D response element-binding protein (VDRE-BP), thereby regulating transcriptional activity of the vitamin D receptor (VDR) bound to 1,25-dihydroxyvitamin D (1,25(OH)2D). In this way hnRNPC1/C2 may act as a coupling factor for 1,25(OH)2D-directed transcription and RNA splicing. Studies using MG63 osteoblastic cells confirmed that 1,25(OH)2D-VDR mediated induction of the gene for the enzyme 24-hydroxylase (CYP24A1), involved CYP24A1-specific chromatin and RNA immunoprecipitation of hnRNPC1/C2. Furthermore, small interfering (siRNA) knockdown of hnRNPC1/C2 in MG63 cells and was associated with dysregulated expression of CYP24A1 and an alternatively spliced form of CYP24A1 (CYP24A1-variant 2). Genome-wide analysis of RNA expression and alternative splicing indicated that dual role of hnRNPC1/C2 in directing 1,25(OH)2D-mediated gene expression is not restricted to the classical VDR-target CYP24A1. Knockdown of hnRNPC1/C2 resulted in 3500 differentially expressed genes (DEG), and treatment with 1,25(OH)2D 324 DEG. A further 87 DEG were only observed in 1,25(OH)2D-treated cells in hnRNPC1/C2 knockdown cells. HnRNPC1/C2 knockdown or 1,25(OH)2D treatment also induced alternative splicing (AS) (5039 and 310 AS events respectively). Combined hnRNPC1/C2 knockdown and 1,25(OH)2D treatment resulted in significant overlap between DEG and AS genes, but this was not observed for 1,25(OH)2D treatment alone. These data indicate that hnRNPC1/C2 can act to couple transcriptional and splicing responses to 1,25(OH)2D by binding to both DNA and RNA. Similar mechanisms may also exist for other members of the hnRNP and steroid receptor family.
Project description:Vitamin D is a pro-hormone that possesses various anticancer effects through diverse mechanisms. The enzyme vitamin D-25-hydroxylase can convert vitamin D into 25-hydroxyvitamin D (25(OH)D) in the liver. 25(OH)D serves as the precursor of 1,25-dihydroxyvitamin D (1,25(OH)2D or calcitriol), which can be transformed into 1,25(OH)2D by the enzyme CYP27B1. CYP27B1 is primarily distributed in the kidneys but can also be found in other tissues, such as the breast and colon. Although 1,25-dihydroxyvitamin D has been the primary object of in vitro studies, emerging evidence suggests that 25(OH)D can also generate anticancer effects by transforming into 1,25(OH)2D in the manner of autocrine and paracrine. Our study aims to investigate the impact of 25(OH)D on breast cancer and its effect on the expression of small RNA in breast cancer cells cultured with 100nM 25(OH)D.
Project description:Vitamin D as a pro-hormone is known to generate anticancer effects by various mechanisms. Vitamin D can be converted to 25-hydroxyvitamin D(25(OH)D) by the enzyme vitamin D-25-hydroxylase in the liver.25(OH)D is the precursor of 1,25‑dihydroxyvitamin D(1,25(OH)2D, also called calcitriol) and can be transformed by the enzyme CYP27B1. The distribution of CYP27B1 is mainly in the kidney, but also in other tissues like breast and colon. So far, research about the anticancer effects of Vitamin D mainly chooses 1,25‑dihydroxyvitamin D as the study object in vitro. However, there is increasing evidence showing that 25(OH)D also can generate anticancer effects by transforming into 1,25(OH)2D in the manner of autocrine and paracrine. Our study aims to explore the influence of 25(OH)D on breast cancer and provides the variation on the expression of RNA in the breast cancer cells cultured with 100nM 25(OH)D.
Project description:We have isolated highly pure testicular extracellular vesicles using a newly established testis dissociation method. We then used shotgun proteomic approach to examine the proteins carried by and expressed on the testicular extracellular vesicles.
Project description:Heterogeneous nuclear ribonucleoprotein (hnRNP) C1/C2 plays a pivotal role in vitamin D receptor (VDR) signaling by acting as a vitamin D response element (VDRE)-binding protein (VDRE-BP). Transcriptional regulation by active 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) involves occupancy of VDRE by VDRE-BP or 1,25(OH)2D3 bound-VDR. This relationship is disrupted by over-expression of VDRE-BP and can cause a form of human hereditary vitamin D-resistant rickets (HVDRR). DNA array analyses using B-cells from an HVDRR patient and matched control defined a sub-cluster of genes where 1,25(OH)2D3-regulated transcription was abrogated by over-expression of VDRE-BP. Amongst these, the DNA-damage-inducible transcript 4 (DDIT4), an inhibitor of mammalian target of rapamycin (mTOR) signaling, was also induced by 1,25(OH)2D3 in human osteoblasts. Chromatin immunoprecipitation using 1,25(OH)2D3-treated osteoblasts confirmed that liganded VDR and VDRE-BP compete for binding to the proximal promoter of the DDIT4 gene in a similar fashion to other known 1,25(OH)2D3-target genes. Treatment of osteoblasts with 1,25(OH)2D3 induced DDIT4 expression and suppressed phosphorylated S6K1T389 protein (a downstream target of mTOR). The functional importance of this for 1,25(OH)2D3 responses in osteoblasts was underlined by the fact that siRNA knockdown of DDIT4 expression suppressed antiproliferative and cell growth responses to 1,25(OH)2D3. These data confirm that VDRE-BP is required for normal 1,25(OH)2D3-mediated transcription and cell function in osteoblasts. Conversely over-expression of VDRE-BP exerts a dominant-negative effect on transcription of 1,25(OH)2D3-target genes. Characterization of VDRE-BP action in 1,25(OH)2D3-treated osteoblasts highlights an entirely novel role for vitamin D as a regulator of mTOR – a known ‘master regulator’ of cell function. We performed gene expression microarray analysis in HVDRR EBV-transformed B-cells and control cells in the presence or absence of vitamin D.
Project description:We analyzed gene expression profiles of human testicular biopsies in men with idiopathic nonobstructive azoospermia. Using new generation oligonucleotide microarray platform GeneChipM-BM-. Human Gene 1.0 ST, we identified genes which could be potential biomarkers of azoospermia and molecular indicators that could determine a particular stage of impaired spermatogenesis. Thus, we shed light on genes which were had so far been weakly characterized and which were had never related to infertility before. These studies also included the comparative analysis of the hierarchical clustering of gene expression profile with histopathological data provided for azoospermic patients. We analyzed 31 testicular biopsy samples with Affymetrix Human Gene 1.0 ST microarrays. 27 of them were obtained from patients with various types of NOA and 4 with normal spermatogenesis.
Project description:1α,25(OH)2VD3 is the most active form of VD3 in animals and it plays an important role in regulating mineral metabolism but also reproduction and immunity. Testes are the main reproductive organs of male mammals. In order to study the effect of 1α,25(OH)2VD3 on early testicular development, 140 weaned 21-day old piglets were selected. The piglets were randomly divided into four groups and were fed a commercial diet supplemented with 0, 1, 2 and 4 μg.kg-1 of 1α,25(OH)2VD3, provided as 1α,25(OH)2VD3-glycosides, 60 days after the start of the experiment, at piglet age 82 days, testes were harvested. The morphology and histology of early testicular development were assessed. In addition, the proteomic TMT/iTRAQ labeling technique was used to analyze the protein profile of the testes in each group. Western blotting was used to verify the target of differentially abundant proteins (DAPs). The results showed that of the identified 132715 peptides, 122755 were specific peptides. 7852 proteins, of which 6573 proteins contain quantitative information. Screening for DAPs was focused on proteins closely related to the regulation of the testicular development such as steroid hormone synthesis, steroid biosynthesis, peroxisome and fatty acid metabolism pathways. These results also indicate that 1α,25(OH)2VD3 is involved in the regulation of early testicular development in piglets. At the same time, these findings provide valuable information for the proteins involved in the regulation of testicular development, and help to better understand the mechanisms of 1α,25(OH)2VD3 in regulating the development of piglets’ testes. Significance: The early development of the testes of breeding boars is closely related to their lifetime fertility. Our research identifies potential signaling pathways and differentially abundant proteins associated with testicular development. These findings indicate that plants containing 1α,25(OH)2VD3-glycosides used as a nutritional ingredient may help early testicular development in breeding boars and reveal the regulatory mechanism of 1α,25(OH)2VD3 on testicular development.