Project description:Systemic lupus erythematosus (SLE) is mediated by a chronic and dysregulated inflammatory response. Interleukin (IL)-17, a proinflammatory cytokine, and T helper (Th)17 cells are associated with chronic autoimmune diseases. We hypothesized that inhibition of IL-17 would decrease the numbers of T cell subsets that function as B-cell helpers, as well as B-cell differentiation into plasma cells and autoantibody expression. The IL-17 level was increased markedly in Roquin<sup>san/san</sup> mice. Loss of IL-17 in Roquin<sup>san/san</sup> mice improved nephritis by downregulating immunoglobulin (Ig)G, IgG1, and IgG2a production. Formation of germinal centers (GCs), and follicular B- and T-cell differentiation was reduced, whereas the number of regulatory T (Treg) cells and immature B cells was increased, by IL-17 deficiency in Roquin<sup>san/san</sup> mice. These results suggest that IL-17 inhibition can ameliorate SLE by inhibiting B-cell differentiation into GCs. Therefore, IL-17-producing Th17 cells show promise as a target for development of novel therapeutics for SLE.
Project description:Roquin is an RNA-binding protein that prevents autoimmunity and inflammation via repression of bound target mRNAs such as inducible costimulator (Icos). When Roquin is absent or mutated (Roquin(san)), Icos is overexpressed in T cells. Here we show that Roquin enhances Dicer-mediated processing of pre-miR-146a. Roquin also directly binds Argonaute2, a central component of the RNA-induced silencing complex, and miR-146a, a microRNA that targets Icos mRNA. In the absence of functional Roquin, miR-146a accumulates in T cells. Its accumulation is not due to increased transcription or processing, rather due to enhanced stability of mature miR-146a. This is associated with decreased 3' end uridylation of the miRNA. Crystallographic studies reveal that Roquin contains a unique HEPN domain and identify the structural basis of the 'san' mutation and Roquin's ability to bind multiple RNAs. Roquin emerges as a protein that can bind Ago2, miRNAs and target mRNAs, to control homeostasis of both RNA species.
Project description:Roquin, an E3 ubiquitin ligase that localizes to cytosolic RNA granules, is involved in regulating mRNA stability and translation. Mice that have a M199R mutation in the Roquin protein (referred to as sanroque or Roquin(san/san) mice) develop autoimmune pathologies, although the extent to which these occur in the intestinal mucosa has not been determined. Here, we demonstrate that Roquin(san/san) mice reproducibly develop intestinal inflammation in the small intestine but not the colon. Similarly, mice generated in our laboratory in which the Roquin gene was disrupted by insertion of a gene trap cassette (Roquin(gt/gt) mice) had small intestinal inflammation that mimicked that of Roquin(san/san) mice. MLN cells in Roquin(san/san) mice consisted of activated proliferating T cells, and had increased numbers of CD44(hi) CD62L(lo) KLRG1(+) short-lived effector cells. Proportionally more small intestinal intraepithelial lymphocytes in Roquin(san/san) mice expressed the ICOS T cell activation marker. Of particular interest, small intestinal lamina propria lymphocytes in Roquin(san/san) mice consisted of a high proportion of Gr-1(+) T cells that included IL-17A(+) cells and CD8(+) IFN-?(+) cells. Extensive cytokine dysregulation resulting in both over-expression and under-expression of chemotactic cytokines occurred in the ileum of Roquin(san/san) mice, the region most prone to the development of inflammation. These findings demonstrate that chronic inflammation ensues in the intestine following Roquin alteration either as a consequence of protein mutation or gene disruption, and they have implications for understanding how small intestinal inflammation is perpetuated in Crohn's disease (CD). Due to the paucity of animal models of CD-like pathophysiology in the small intestine, and because the primary gene/protein defects of the Roquin animal systems used here are well-defined, it will be possible to further elucidate the underlying genetic and molecular mechanisms that drive the disease process.
Project description:Roquin, an E3 ligase, is involved in curtailing autoimmune pathology as seen from studies using mice with mutated (Rc3h1(san/san)) or disrupted (Rc3h1(gt/gt)) Rc3h1 gene. The extent to which intestinal immunopathology is caused by insufficient Roquin expression in the immune system, or by Roquin impairment in non-hematopoietic cells, has not been determined. Using bone marrow cells from Rc3h1(gt/gt) mice transferred into irradiated normal mice (Rc3h1(gt/gt) → NL chimeras), we show that inflammation developed in the small intestine, kidney, lung, liver, and spleen. Proinflammatory cytokine levels were elevated in lamina propria lymphocytes (LPLs). Inflammation in the liver was accompanied by areas of hepatocyte apoptosis. Lung inflammation consisted of an influx of both T cells and B cells. Small intestinal LPLs had increased numbers of CD44(hi), CD62L(lo), KLRG1(+), ICOS(+) short-lived effector cells, indicating an influx of activated T cells. Following oral infection with L. monocytogenes, Rc3h1(gt/gt) → NL chimeras had more liver pathology and greater numbers of bacteria in the Peyer's patches than NL → NL chimeras. These findings demonstrate that small intestinal inflammation in Rc3h1(san/san) and Rc3h1(gt/gt) mice is due to a failure of Roquin expression in the immune system and not to insufficient systemic Roquin expression.
Project description:IL-10(-/-) mice, an animal model of Th1-mediated inflammatory bowel disease, were screened for the expression of 600 microRNAs (miRNAs) using colonic tissues and PBLs from animals having either mild inflammation or severe intestinal inflammation. The development of colonic inflammation in IL-10(-/-) mice was accompanied by upregulation in the expression of 10 miRNAs (miR-19a, miR-21, miR-31, miR-101, miR-223, miR-326, miR-142-3p, miR-142-5p, miR-146a, and miR-155). Notably, the expression of all of these miRNAs plus miR-375 was elevated in PBLs of IL-10(-/-) mice at a time when colonic inflammation was minimal, suggesting that changes in specific miRNAs in circulating leukocytes may be harbingers of ensuing colonic pathology. In vitro exposure of colonic intraepithelial lymphocytes to IL-10 resulted in downregulation of miR-19a, miR-21, miR-31, miR-101, miR-223, and miR-155. Interestingly, unlike IL-10(-/-) mice, changes in miRNAs in PBL of dextran sulfate sodium-treated mice were minimal but selectively elevated in the colon after pathology was severe. We further show that miR-223 is a negative regulator of the Roquin ubiquitin ligase, Roquin curtails IL-17A synthesis, and the 3' untranslated region of Roquin is a target for miR-223, thus defining a molecular pathway by which IL-10 modulates IL-17-mediated inflammation. To identify additional miRNAs that may be involved in the regulation of Roquin, transcriptome analysis was done using cDNAs from HeLa cells transfected with 90 miRNA mimics. Twenty-six miRNAs were identified as potential negative regulators of Roquin, thus demonstrating functional complexity in gene expression regulation by miRNAs.
Project description:MicroRNAs (miRNAs) are important post-transcriptional regulators of gene expression that act by degrading their RNA targets or by repressing the translation of messenger RNAs (mRNAs). Initially thought to primarily target the 3' untranslated region (3'UTR) of mRNAs, miRNAs have since been shown to also target the 5'UTR and coding sequence (CDS). In this work, we focus on the post-transcriptional regulation of the BRCA1 gene, a major tumor suppressor and regulator of double-stranded break DNA repair and show that its mRNA is targeted by many members of the miR-15/107 group at a site located within the CDS. Ectopic expression of these miRNAs across a panel of nine cell lines demonstrated widespread suppression of BRCA1 mRNA levels. Additionally, by cloning a putative target site from BRCA1's amino acid CDS into a luciferase reporter plasmid we confirmed the direct interaction of these miRNAs with this BRCA1 target. We also examined the relationship between ectopic expression of these targeting miRNAs and BRCA1 protein levels in immortalized pancreatic epithelium (hTERT-HPNE), colorectal adenocarcinoma (HCT-116) and pancreatic adenocarcinoma (MIA PaCa-2) cell lines and found protein abundance to be variably regulated in a cell-type specific manner that was not necessarily concordant with mRNA transcript availability. Our findings reveal a previously unrecognized aspect of BRCA1's miRNA-mediated post-transcriptional regulation, namely the targeting of its amino acid coding region by the miR-15/107 group of miRNAs. The resulting regulation is apparently complex and cell-specific, an observation that may have implications for BRCA1-mediated DNA repair across tissue types.
Project description:Background:Dangguijagyag-san, also known as Dangguishaoyao-san in Chinese and Toki-shakuyakui-san in Japanese, has been frequently used to treat symptoms associated with dysmenorrhea. The purpose of this trial is to evaluate the efficacy and safety of the herbal medicine, Dangguijagyag-san, relative to those of active control, Gamisoyo-san, and a placebo control for primary dysmenorrhea. Methods:This protocol details a randomized, double-blind, parallel-group, multi-center, investigator-initiated, controlled trial evaluating treatment of primary dysmenorrhea. Two hundred and forty participants will be randomly divided into one of three groups: 1) the Dangguijagyag-san experimental group (EG) (n = 105), 2) the Gamisoyo-san active control group (ACG) (n = 30), and 3) the placebo control group (PCG) (n = 105). The interventions will be administered for two menstrual cycles, and the follow-up will be carried out for the following six menstrual cycles. The primary outcomes are difference in response rates between the EG and the ACG (non-inferiority comparison) and difference in changes from baseline in average pain intensity measured by the visual analogue scale between the EG and PCG (superiority comparison). The secondary outcomes are pain scores derived from pain assessment tools (verbal multidimensional scoring system, retrospective symptom scale, and short form McGill pain questionnaire), dosage of analgesics, pattern diagnosis questionnaires, and short form 36 health survey. Adverse events and vital signs will be checked at every visit, and laboratory tests will be performed for safety evaluation. Discussion:The results of this clinical trial will offer evidence for the efficacy and safety of Dangguijagyag-san for primary dysmenorrhea. Trial registration:Clinical Research Information Service of Korea: KCT0003005.
Project description:miRNAs expression of tumor sample of mexican patients with breast cancer. Samples obtained from the Hospital San Jose Tec de Monterrey. The experiments were with one color per patient, miRNAs expression profile is from a tumor sample of mexican patients with breast cancer.
Project description:To determine the miRNA expression pattern in RBCs, we isolated miRNA from RBCs of healthy blood donors, who were homozygous or heterozygous carriers of different AB0 blood groups. We first analyzed miRNA expression by microarray chip analysis. In average, we found 873 miRNAs to be present in RBCs with a partially differential expression pattern depending on the blood group genotype. 148 out of these 873 miRNAs were significantly up- or downregulated in RBCs of blood group 0 and of heterozygous genotypes, as compared to homozygous genotypes. For each blood group genotype erythrocyte concentrates of three different donors were used for microarray analysis.
Project description:Qian-Zheng-San, a traditional Chinese prescription consisting of Typhonii Rhizoma, Bombyx Batryticatus, Scorpio, has been found to play an active therapeutic role in central nervous system diseases. However, it is unclear whether Qian-Zheng-San has therapeutic value for peripheral nerve injury. Therefore, we used Sprague-Dawley rats to investigate this. A sciatic nerve crush injury model was induced by clamping the right sciatic nerve. Subsequently, rats in the treatment group were administered 2 mL Qian-Zheng-San (1.75 g/mL) daily as systemic therapy for 1, 2, 4, or 8 weeks. Rats in the control group were not administered Qian-Zheng-San. Rats in sham group did not undergo surgery and systemic therapy. Footprint analysis was used to assess nerve motor function. Electrophysiological experiments were used to detect nerve conduction function. Immunofluorescence staining was used to assess axon counts and morphological analysis. Immunohistochemical staining was used to observe myelin regeneration of the sciatic nerve and the number of motoneurons in the anterior horn of the spinal cord. At 2 and 4 weeks postoperatively, the sciatic nerve function index, nerve conduction velocity, the number of distant regenerated axons and the axon diameter of the sciatic nerve increased in the Qian-Zheng-San treatment group compared with the control group. At 2 weeks postoperatively, nerve fiber diameter, myelin thickness, and the number of motor neurons in the lumbar spinal cord anterior horn increased in the Qian-Zheng-San treatment group compared with the control group. These results indicate that Qian-Zheng-San has a positive effect on peripheral nerve regeneration.