MicroRNA effect on salivary flow of Sjogren's syndrome patients

MicroRNA对干燥综合征患者唾液流量的影响

• 批准号: 8553353
• 负责人: Ilias Alevizos
• 金额: $ 77.12万
• 依托单位: NATIONAL INSTITUTE OF DENTAL & CRANIOFACIAL RESEARCH
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8553353
• 关键词: 3' Untranslated Regions; Acinar Cell; Affect; Algorithms; Binding; Bioinformatics; Biological Assay; Biopsy; Calcium; Calcium Channel; Cell physiology; Cells; Chronic; Code; Cytosol; Ductal Epithelial Cell; Endoplasmic Reticulum; Epithelial Cells; Event; Fluorescence; Functional disorder; Genes; Goals; Human; Human Herpesvirus 4; Immunofluorescence Immunologic; Laboratories; Luciferases; Measurement; Measures; Mediating; Messenger RNA; MicroRNAs; Minor salivary gland structure; Molecular; Mus; Pathway interactions; Patients; Plasmids; Process; Proteins; Reporting; Role; STIM1 gene; Salivary; Salivary Glands; Sjogren' s Syndrome; Testing; Thapsigargin; Transcript; Transfection; Translations; Viral; analog; attenuation; cell type; healthy volunteer; member; receptor; saliva secretion; sensor; systemic autoimmune disease; viral DNA

Loss of secretory function of salivary glands is one of the most important functional effects of Sjogren's syndrome (SS), a chronic systemic autoimmune disease. Previous studies have reported the presence of Epstein Barr Virus (EBV) DNA in salivary glands of SS patients. MicroRNA profiling studies in our laboratory have identified an EBV microRNA (ebv-mir-BART13) as significantly over-expressed in minor salivary gland biopsies of SS patients, when compared to the expression in minor salivary glands from healthy volunteers. Using the RNA22 target prediction algorithm, we identified STIM1 3'UTR and coding sequence as targets of ebv-mir-BART13. STIM1, is a protein that has recently been identified as a critical molecular component of the calcium release-activated calcium current (CRAC) channels. Store-operated calcium entry (SOCE) and CRAC are critical events for the replenishment of intracellular calcium stores and have indispensable roles in various cellular functions, including salivary secretion. SOCE is activated by the depletion of calcium in the endoplasmic reticulum (ER). STIM1, is suggested to be the ER-Ca2+ sensor protein regulating SOCE in a number of different cell types. Attenuation of SOCE current underlies salivary gland dysfunction in mice lacking transient receptor potential 1 (TRPC1). We hypothesized that ebv-miR-Bart-13 may contribute to the salivary gland dysfunction present in SS by downregulating STIM-1 and decreasing calcium influx in salivary gland cells. To test this hypothesis, HSG -human submandibular salivary gland cells- were transfected for 24 and 48hrs with ebv-miR-BART13 analog and the mRNA and protein levels of STIM1 were measured. The mRNA level was not affected by the presence of this viral miRNA. However, the protein level was dramatically decreased both 24 and 48hrs after transfection, suggesting that the ebv-mir-Bart13 exerts its effects on STIM1 not by degrading its transcript, but by repressing STIM1 translation. To examine if the effect on STIM1 protein dowregulation was mediated by binding on the 3UTR, we co-transfected HSG cells with the STIM1 3UTR cloned sequence within a luciferase plasmid along with EBV-mir-BART13 mimics, and antagonists. A 40% decrease in the luciferase expression was observed with the mimic and plasmid co-transfection, confirming that this viral microRNA binds to the 3UTR of STIM1. To examine if binding also occurs on the coding sequence of the STIM1 transcript, we co-transfected HSG cells with a YFP plasmid containing STIM1 coding sequence with ebv-mir-BART13 mimics, and antagonists. Through immunofluorescence we observed a distinct decrease in STIM1 fluorescence levels in the EBV-mir-BART13 mimic transfected cells compared to controls. Functional Ca2+ assays through Thapsigargin-mediated depletion of the endoplasmic reticulum calcium in the ebv-mir-BART13 transfected cells showed a significant delay in the influx of calcium in the cytosol compared to controls. Together, these functional measurements suggest that the presence of ebv-miR-BART13 in the salivary glands of Sjogren's syndrome patients might be, at least partially, responsible for the salivary gland dysfunction in those patients, by reducing the expression of STIM1, a critical member of the saliva secretion mechanism. Besides ebv-mir-BART13, we identified several more microRNAs that are differentially expressed in the minor salivary glands of SS patients. Through extensive bioinformatics analysis we have identified genes known to be involved in salivary gland function that might be differentially expressed in SS salivary glands and we are in the process of validating their altered expression.

干燥综合征（SS）是一种慢性全身性自身免疫性疾病，唾液腺分泌功能的丧失是其最重要的功能效应之一。先前的研究已经报道了SS患者唾液腺中存在Epstein巴尔病毒（EBV）DNA。我们实验室的microRNA分析研究已经鉴定了一种EBV microRNA（ebv-mir-BART 13），当与健康志愿者的小唾液腺中的表达相比时，其在SS患者的小唾液腺活检中显著过表达。 使用RNA 22靶标预测算法，我们鉴定了STIM 1 3 'UTR和编码序列作为ebv-mir-BART 13的靶标。STIM 1是最近被鉴定为钙释放激活的钙电流（CRAC）通道的关键分子组分的蛋白质。钙库操纵的钙进入（SOCE）和CRAC是细胞内钙库补充的关键事件，在包括唾液分泌在内的各种细胞功能中具有不可或缺的作用。SOCE通过内质网（ER）中钙的消耗而激活。STIM 1被认为是在许多不同细胞类型中调节SOCE的ER-Ca 2+传感器蛋白。 SOCE电流衰减是瞬时受体电位1（TRPC 1）缺失小鼠唾液腺功能障碍的基础。我们推测ebv-miR-Bart-13可能通过下调STIM-1和减少唾液腺细胞中的钙内流而导致SS中存在的唾液腺功能障碍。 为了验证这一假设，用ebv-miR-BART 13类似物转染HSG -人下颂唾液腺细胞24和48小时，并测量STIM 1的mRNA和蛋白水平。mRNA水平不受该病毒miRNA存在的影响。然而，在转染后24和48小时，蛋白水平显著降低，这表明ebv-mir-Bart 13对STIM 1的作用不是通过降解其转录物，而是通过抑制STIM 1的翻译。 为了检测对STIM 1蛋白下调的影响是否是通过结合在3UTR上介导的，我们将荧光素酶质粒内的STIM 1 3UTR克隆序列沿着EBV-mir-BART 13模拟物和拮抗剂一起共转染HSG细胞。在模拟物和质粒共转染的情况下观察到荧光素酶表达降低40%，证实该病毒microRNA结合STIM 1的3UTR。为了检查结合是否也发生在STIM 1转录物的编码序列上，我们用含有STIM 1编码序列的YFP质粒与ebv-mir-BART 13模拟物和拮抗剂共转染HSG细胞。通过免疫荧光，我们观察到与对照相比，在EBV-mir-BART 13模拟物转染的细胞中STIM 1荧光水平明显降低。 功能性Ca 2+测定通过Thapsiglavin介导的消耗内质网钙的ebv-mir-BART 13转染的细胞中，显示了显着延迟钙流入细胞质相比，控制。 总之，这些功能测量表明，干燥综合征患者唾液腺中ebv-miR-BART 13的存在可能至少部分地导致这些患者的唾液腺功能障碍，通过减少STIM 1的表达，STIM 1是唾液分泌机制的关键成员。 除了ebv-mir-BART 13，我们还鉴定了几种在SS患者的小唾液腺中差异表达的microRNA。通过广泛的生物信息学分析，我们已经确定了已知参与唾液腺功能的基因，这些基因可能在SS唾液腺中差异表达，我们正在验证其改变的表达。