MicroRNA expression profile from medullary thymic epithelial cells - mTECs - from pre-diabetic NOD mice
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ABSTRACT: Due to high sequence identity between human and M. musculus microRNAs, we used in this study human microRNA microarrays to determine microRNA expression profiles of medullary thymic epithelial cells (mTECs) from thymus of pre-diabetic NOD mice.
Project description:The oligo microarrays were used to determine mRNA expression profiles of medullary thymic epithelial cells (mTECs) isolated from thymus of pre-diabetic NOD mice.
Project description:The microRNA oligo microarrays were used to determine expression profiles of peripheral blood mononuclear cells from type 2 diabetes mellitus (T2DM) patients, aiming the identification of possible disease related MicroRNAs.
Project description:The oligo microarrays were used to determine microRNA expression profiles of medullary thymic epithelial cells (mTECs) submitted of in vitro Aire Knockdown (siRNA silencing).
Project description:The medullary thymic epithelial cells (mTECs) express virtually all autoantigens of the body. This phenomenon was then termed promiscuous gene expression (PGE). A large set of autoantigen genes (but not all) is controlled by the transcriptional modulator Autoimmune regulator (Aire) in mTECs. These autoantigens represent all tissues and organs in the thymus and it is implicated in the negative selection of autoreactive thymocytes, and consequantly preventing autoreactive autoimmune reactions and autoimmune diseases (e.g. type 1 diabetes mellitus, systemic lupus erythematosus). Thus, we are looking at gene expression in thse cells because it is very important to better understand the molecular basis of central immune tolerance to normal tissues and organs. The aim of this study is to evaluate the possible link between the expression of the transcriptional regulator Aire, the genetic background of mouse strains and the promiscuous gene expression in mTECs.
Project description:Non-obese diabetic (NOD) mice feature pancreatic infiltration of autoreactive T lymphocytes as early as 1 month of age, which destruct insulin-producing beta-cells to finally emerge autoimmune diabetes mellitus (T1D) within 8 months. In view, we hypothesized that during the development of T1D, transcriptional modulation of immune reactivity genes may occur as thymocytes mature toward peripheral T lymphocytes. Transcriptome of thymocytes and peripheral CD3+ T lymphocytes from pre-diabetic or diabetic mice analyzed through microarray hybridizations allowed observation of 3,586 differentially expressed genes. Hierarchical clustering grouped mice according to age/T1D onset and genes to their ontology. Transcriptional activity of thymocytes toward peripheral T lymphocytes unraveled sequential participation of genes involved with CD4+/CD8+ T cell differentiation (Themis), tolerance induction (Foxp3), apoptosis (Fasl) to soon after T cell activation (IL4), while the emergence of T1D coincided with the expression of cytotoxicity (Crtam) and inflammatory response genes (Tlr) by peripheral T lymphocytes.
Project description:The oligo micoarrays were used to determine gene expression profiles of peripheral blood mononuclear cells from gestational diabetes mellitus (GDM) patients.
Project description:Cortical 1.4C18 and medullary 3.10 TEC lines (mTEC) were cultured in 10% fetal bovine serum-supplemented RPMI 1640 medium at 37 ?C in a 5% CO2 atmosphere. Confluent cultures of 3.10 mTEC cell line were cultured during 72 h in RPMI medium as above mentioned and total RNA was extracted using the mirVana kitM-. (Ambion, Austin, TX, USA).
Project description:Small interfering RNA (siRNA) was used to knockdown Autoimmune regulator(Aire) gene by in vivo electroporation of the thymus of BALB-c mice. In This set of data we include control and Aire-silenced mTEC cells isolated from thymus of BALB-c mice.
Project description:We used the TriFectaTM (IDT, Integrated DNA Technologies, Coralville, IA, USA) anti-Aire RNAi sequence (GGAUUCUCUUUAAGGACUACAAUCTAGAUUGUAGUCCUUAAAGAGAAUCCUC) in order to silencing Aire mRNA. Confluent cultures of 3.10 mTEC cell line were transfected with 10nM of anti-Aire siRNA using Hiperfect reagent (Qiagen, GmbH, Hilden, Germany) following manufacturerM-^Rs instructions. After transfection, cells were cultured during 24 h in RPMI medium as above mentioned and total RNA was extracted using the mirVana kitM-. (Ambion, Austin, TX, USA), which served as template for cDNA synthesis.<br><br>Gene knockdown was confirmed by quantitative reverse transcription PCR (qRT-PCR) using the primers 5_-GCAACTCTGGCCTCAAAGAG-3_ forward and 5_-GGTCTGAATTCCGTTTCCAA-3_ reverse, which allowed amplification<br><br>of a 120 bp PCR product corresponding to a segment of the Aire cDNA. The cDNA samples were prepared using Superscript II<br><br>reverse transcriptase (Invitrogen Corporation, Carlsbad, CA, USA) enzyme, as recommended. Expression of the above mentioned genes was quantified using a 7500 Real Time PCR System (Applied Biosystems)
Project description:Female NOD mice were born in specific pathogen free (SPF) conditions at the CEMIB-UNICAMP animal facility (University of Campinas, S�o Paulo, Brazil) and maintained in SPF mini-isolators in our laboratory at the University of S�o Paulo, Campus of Ribeir�o Preto, Brazil. We studied both pre-diabetic (8�2 week-old) and diabetic (20�2 week-old) animals. Diabetes was confirmed by blood glucose levels (?250 mg glucose/dL) using the Accu Check � Active Kit (Roche Diagn�stica Brasil, S�o Paulo, Brazil). The thymic stroma was separated from the whole thymus. The mTEC 3.10 medullary thymic epithelial cell line was established from C57BL/6 mice, and the original medullary phenotype was confirmed by immunostaining with anti-cytokeratin monoclonal antibodies. In addition, the CD80+ phenotype was confirmed using fluorescent activated cell sorting (FACS) analysis. Cells were cultured in 10% fetal bovine serum-supplemented RPMI 1640 medium at 37oC and 5% CO2. Total RNA was extracted from 1x107 stromal cells (from pre-diabetic and diabetic animals) and 1x107 mTEC 3.10 cells using Trizol� Reagent and following the manufacturer�s instructions (Invitrogen, Carlsbad, CA, USA). RNA preparations were confirmed to be free of proteins and phenol using UV spectrophotometry and the state of degradation was assessed using agarose gel electrophoresis (ethidium bromide staining).