MicroRNA in autoimmunity and autoimmune diseases

MicroRNAs (miRNAs) are small conserved non-coding RNA molecules that post-transcriptionally regulate gene expression by targeting the 3′ untranslated region (UTR) of specific messenger RNAs (mRNAs) for degradation or translational repression. miRNA-mediated gene regulation is critical for normal cellular functions such as the cell cycle, differentiation, and apoptosis, and as much as one-third of human mRNAs may be miRNA targets. Emerging evidence has demonstrated that miRNAs play a vital role in the regulation of immunological functions and the prevention of autoimmunity. Here we review the many newly discovered roles of miRNA regulation in immune functions and in the development of autoimmunity and autoimmune disease. Specifically, we discuss the involvement of miRNA regulation in innate and adaptive immune responses, immune cell development, T regulatory cell stability and function, and differential miRNA expression in rheumatoid arthritis and systemic lupus erythematosus

MiR-146a Upregulation of Phagocytosis in Human Macrophages

Sjögrens Syndrome (SjS) is an autoimmune disease that attacks exocrine glands such as salivary and lacrimal glands resulting in severe dryness of the mouth and eyes. Previous studies have linked increased microRNA-146a (miR-146a) expression in peripheral blood mononuclear cells in SjS patients compared to healthy controls. MicroRNAs (miRNAs), small non-coding RNA molecules that post-transcriptionally regulate gene expression, are known to play key regulatory roles in immune responses and have been implicated in a growing number of autoimmune disorders. Further investigation into the role of increased miR-146a expression in SjS revealed links to several immune functions including phagocytosis.Our goal was to further examine the relationship between miR-146a expression and the rate of phagocytosis in human macrophages by using apoptotic human cells as a phagocytic target. We hypothesized that upregulation of miR-146a would increase phagocytic activity of differentiated THP-1 human monocytes. To quantify phagocytic activity, a pH-sensitive fluorescent dye (pHrodo) was used to indicate the E. coli or apoptotic Jurkats that had been phagocytosed. THP-1 cells were transfected with miR-146a and differentiated into macrophages. Phagocytic activity was observed by incubating fluorescently labeled E.coli or apoptotic Jurkat cells with miR-146a transfected and mock transfected THP-1 cells for 2-4 hours. Fluorescence intensity was quantified using a fluorescent plate reader (E. coli) and microscopy (apoptotic Jurkats). MiR-146a-transfected THP-1 cells exhibited significantly increased phagocytic activity of fluorescently labeled E. coli (P\u3c0.001) and apoptotic Jurkats. Knockdown of TRAF6, a gene target of miR-146a, did not impact the phagocytic activity. MiR-146a appears to upregulate phagocytic activity in human THP-1 cells through an unknown mechanism. Further studies are in progress to determine the mechanism by which miR-146a upregulates phagocytosis

Presence of \u3cem\u3ePorphyromonas gingivalis\u3c/em\u3e in gingival squamous cell carcinoma

Periodontal disease has been recently linked to a variety of systemic conditions such as diabetes, cardiovascular disease, preterm delivery, and oral cancer. The most common bacteria associated with periodontal disease, Porphyromonas gingivalis (P. gingivalis) has not yet been studied in the malignant gingival tissues. The objective of this study was to investigate the presence of P. gingivalis in specimens from squamous cell carcinoma patients. We have performed immunohistochemical staining to investigate the presence of P. gingivalis and Streptococcus gordonii (S. gordonii), a non invasive oral bacteria, in paraffin embedded samples of gingival squamous cell carcinoma (n=10) and normal gingiva (n=5). Staining for P. gingivalis revealed the presence of the bacteria in normal gingival tissues and gingival carcinoma, with higher levels (more than 33%,P\u3c0.05) detected in the carcinoma samples. The staining intensity was also significantly enhanced in the malignant tissue by 2 folds (P\u3c0.023) compared to specimens stained for the non‐invasive S. gordonii. P. gingivalis is abundantly present in malignant oral epithelium suggesting a potential association of the bacteria with gingival squamous cell carcinoma

Effect of Metformin on miR-146a Expression

Sjögren’s Syndrome (SjS) is an autoimmune disorder that affects secretory glands in the human body, restricting their function and causing extreme dryness in areas like the mouth and eyes. miR-146a is an anti-inflammatory microRNA that targets the NFκB activation pathway. Previous studies have shown that SjS patients have increased miR-146a expression, despite having high levels of inflammation. The objective of this study was to investigate whether metformin, a diabetes drug with a wide variety of effects and potential functions, reduces levels of miR-146a expression. Metformin is known to reduce inflammation by inhibiting the activation of NFκB. THP-1 human monocytes were treated with various concentrations of metformin ranging from 12.5uM to 200uM. The cells were treated for 24 hours before total RNA was isolated, and qRT-PCR was utilized to compare miR-146a expression in metformin-treated versus untreated cells. Our results showed a dose-dependent decrease of miR-146a expression in the presence of metformin. These results are reasonable since miR-146a expression is dependent on NFκB activation, and metformin is known to inhibit the activation of NFκB. Further studies will investigate metformin’s ability to suppress inflammation in varying conditions

Migration Frustrations of miR-146a Regulation

The autoimmune disease, Sjögren’s Syndrome (SS), causes the degradation of salivary and lacrimal glands due to an influx of immune cells. In previous studies, a significant increase in miR-146a was observed in the peripheral blood mononuclear cells of SS patients. Since immune cell infiltration is critical in SS pathogenesis, the following research examines the effect of miR-146a on cell migration. We hypothesize that transfecting THP-1 human monocytes with synthetic miR-146a will downregulate migration of the monocytes based on other studies stating that miR-146a downregulates migration in vivo. In order to execute our experiment, we transfected THP-1 cells with synthetic miR-146a and incubated the monocytes for 3 days. In the migration assay, the cells were transferred to a semipermeable membrane and MCP-1 was introduced as a chemoattractant. qPCR was also used to confirm the success of the transfection. When compared to mock-transfected and negative control cells, a significant increase of migration was observed in the THP-1 transfected cells (p value = 0.002 and 0.01, respectively). The qPCR also revealed an upregulation of miR-146a expression. In previous studies miR-146a directly inhibited TRAF6. Considering this evidence, we decided to knockdown TRAF6 with siRNA to observe the migrational effect. Our preliminary data shows that knockdown of TRAF6 decreases migration. Further experimentation must be conducted in order to ascertain the signaling pathway of miR-146a in migration, since it appears that miR-146a does not affect migration through TRAF6. Our data suggests that the original hypothesis was incorrect and that miR-146a stimulates migration of THP-1 cells through an undetermined mechanism

Detection of the argonaute protein Ago2 and microRNAs in the RNA induced silencing complex (RISC) using a monoclonal antibody

MicroRNAs (miRNAs) are short RNA molecules responsible for post-transcriptional gene silencing by the degradation or translational inhibition of their target messenger RNAs (mRNAs). This process of gene silencing, known as RNA interference (RNAi), is mediated by highly conserved Argonaute (Ago) proteins which are the key components of the RNA induced silencing complex (RISC). In humans, Ago2 is responsible for the endonuclease cleavage of targeted mRNA and it interacts with the mRNAbinding protein GW182, which is a marker for cytoplasmic foci referred to as GW bodies (GWBs). We demonstrated that the antiAgo2 monoclonal antibody 4F9 recognized GWBs in a cell cycle dependent manner and was capable of capturing miRNAs associated with Ago2. Since Ago2 protein is the effector protein of RNAi, anti-Ago2 monoclonal antibody may be useful in capturing functional miRNAs

Expression of miR-145 and miR-449 in U87 Glioblastoma Cells

MicroRNAs are known to play a critical oncogenic role in glioblastoma. Several miRNAs have been shown to play roles in growth and cell cycle control in glioblastoma, and have potential as diagnostic markers or as therapeutic targets. miRNA-145 is overexpressed in glioblastoma and is thought to downregulate srGAP1 (SLIT-ROBO Rho GTPase-activating protein1), which promotes an invasive phenotype. This is in contrast to miRNA-145’s proposed tumor-supressive role in many other cancers (1). miRNA-449 is thought to be a tumor suppressor that interrupts the cell cycle and induces apoptosis via suppression of E2F1, CCND1, and GPR158. Therefore it is highly downregulated in many tumor cells (2,3). Both miRNAs-145 and -449 are the topic of continued inquiry in understanding their expression and their molecular targets in glioma cells. Here we attempt to establish the baseline expression of miRNA-145 and miRNA-449 in the U87 cell line using RT-PCR. Weekly passaging of cells was carried out, followed by RNA isolation and RT-PCR. Baseline average concentrations were established for miRNA-145 and miRNA-449 using a set of serial dilutions and a standard curve. Further experimentation will be required to establish a baseline concentration for these miRNAs in healthy glial cells

Upregulated miR-146a expression in peripheral blood mononuclear cells from rheumatoid arthritis patients

Introduction MicroRNAs are small noncoding RNA molecules that negatively regulate gene expression via degradation or translational repression of their targeted mRNAs. It is known that aberrant microRNA expression can play important roles in cancer, but the role of microRNAs in autoimmune diseases is only beginning to emerge. In this study, the expression of selected microRNAs is examined in rheumatoid arthritis. Methods Total RNA was isolated from peripheral blood mononuclear cells obtained from patients with rheumatoid arthritis, and healthy and disease control individuals, and the expression of miR-146a, miR-155, miR-132, miR-16, and microRNA let-7a was analyzed using quantitative real-time PCR. Results Rheumatoid arthritis peripheral blood mononuclear cells exhibited between 1.8-fold and 2.6-fold increases in miR-146a, miR-155, miR-132, and miR-16 expression, whereas let-7a expression was not significantly different compared with healthy control individuals. In addition, two targets of miR-146a, namely tumor necrosis factor receptor-associated factor 6 (TRAF6) and IL-1 receptor-associated kinase 1 (IRAK-1), were similarly expressed between rheumatoid arthritis patients and control individuals, despite increased expression of miR-146a in patients with rheumatoid arthritis. Repression of TRAF6 and/or IRAK-1 in THP-1 cells resulted in up to an 86% reduction in tumor necrosis factor-α production, implicating that normal miR-146a function is critical for the regulation of tumor necrosis factor-α production. Conclusions Recent studies have shown that synovial tissue and synovial fibroblasts from patients with rheumatoid arthritis exhibit increased expression of certain microRNAs. Our data thus demonstrate that microRNA expression in rheumatoid arthritis peripheral blood mononuclear cells mimics that of synovial tissue/fibroblasts. The increased microRNA expression in rheumatoid arthritis patients is potentially useful as a marker for disease diagnosis, progression, or treatment efficacy, but this will require confirmation using a large and well defined cohort. Our data also suggest a possible mechanism contributing to rheumatoid arthritis pathogenesis, whereby miR-146a expression is increased but unable to properly function, leading to prolonged tumor necrosis factor-α production in patients with rheumatoid arthritis

Genetic Variation in Concentration of the 33-mer Protein Subcomponent in Wheat

Celiac Disease is a hypersensitive response to gluten caused by HLA-DQ2 or HLA-DQ8 T-cell presentation, initiating destruction of intestinal epithelial cells. Currently, the only remedy for those suffering from celiac disease is elimination of all gluten from the diet. Studies indicate that an indigestible fragment of the gluten molecule, alpha-gliadin subcomponent 33-mer, rich in proline and glutamine, is responsible for the hypersensitivity response. Determination of 33-mer concentration in wheat lines could be beneficial to future development of wheat lines with reduced 33-mer concentration. Protein from wheat flour was extracted and subjected to ELISA techniques in order to quantify the concentration of 33-mer. A technique that quantifies the concentration of 33-mer is a necessary first step for future research efforts focused on identification and development of wheat lines with reduced concentrations of 33-mer. It is possible that wheat with reduced 33-mer may be suitable for consumption by individuals with celiac disease