Regulation of T-bet expression in TH17 cells by microRNAs

microRNA 对 TH17 细胞中 T-bet 表达的调节

• 批准号: 8553181
• 负责人: Vanja Lazarevic
• 金额: $ 36.56万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 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/8553181
• 关键词: Address; Affect; Animals; Area; Autoimmunity; Binding; Binding Sites; Bioinformatics; CD4 Positive T Lymphocytes; Cells; Complex; Control Animal; Data; Development; Frequencies; Gene Expression; Genes; Goals; Immunity; Immunology; Interferon Type II; Interleukin-17; Investigation; Link; MicroRNAs; Mutate; Nature; Neuraxis; Pathway interactions; Process; Regulation; Role; Signal Pathway; Surface; Untranslated Regions; human DICER1 protein; interleukin-23; pathogen; response

A robust TH17 response is detected in the CNS (central nervous system) of T-bet deficient animals, which is stronger in frequency and magnitude than in control animals, suggesting that T-bet acts as a suppressor TH17 lineage commitment. We have shown that T-bet silences the expression of RORgt in developing TH17 cells by blocking the transcriptional activity of Runx1, a positive regulator of RORgt gene expression. Runx1 also acts as a co-activator and together with RORgt induces the expression of the Il17a and Il17f genes. Therefore, the binding of T-bet to Runx1 will deplete the pool of free Runx1 available for the formation of transcriptionally active Runx1/RORgt complexes in TH17 cells. These data suggest that if a naive CD4+ is to be differentiated into a TH17 cell, it has to repress T-bet expression. The mechanism by which T-bet is silenced in developing TH17 cells remains obscure. Two recent studies have demonstrated that T-bet expression in TH1 cells is regulated post-transcriptionally by the microRNA miR-29b [Immunity (2011) 35:169-181; Nature Immunology (2011) 12:861-869]. Our preliminary data using Dicer-deficient TH17 cells suggest that T-bet expression is negatively regulated by microRNAs in this TH subset. The goal of this project was to identify which microRNA is responsible for suppressing T-bet expression in TH17 cells. We performed microRNA microarrays on TH cells that express T-bet (such as TH0 and IL-23 treated TH17 cells) and in TH17 cells, which do not express T-bet. Differential analysis revealed several microRNAs that were expressed at high levels in TH17 cells and diminished in TH cells expressing T-bet. Furthermore, bioinformatics analysis revealed the presence of binding sites in the T-bet 3?-UTR specific for these microRNAs. Collectively, these results suggest that the selected microRNAs are good candidates for the silencing of T-bet expression during TH17 differentiation. To directly address this question, we will over-express the candidate microRNAs in TH1 cells, which normally have high levels of T-bet, and investigate whether the presence of these microRNAs in TH1 cells have a negative effect on T-bet expression. If this turns out to be the case, we will silence these microRNAs in TH17 cells and investigate whether this manipulation will affect TH17 differentiation or T-bet expression in IL-17A producing cells. In addition, we will mutate specific microRNA binding sites in the T-bet 3?-UTR region and determine whether these microRNAs act as direct regulators of T-bet expression, or they act indirectly by affecting signaling pathways that result in T-bet induction (such as IFN-g signaling pathway, for example) The results from this study will reveal the basic understanding of the regulatory mechanisms that act to constrain the T-bet specific pathway that promotes the development of the opposing TH1 lineage.

在T-bet缺乏动物的中枢神经系统（中枢神经系统）中检测到了强大的Th17反应，该动物的频率和幅度比对照动物更强，这表明T-bet充当抑制器Th17谱系承诺。我们已经表明，T-bet通过阻止RORGT基因表达的阳性调节剂Runx1的转录活性来使RORGT在开发Th17细胞中的表达保持沉默。 Runx1还充当共激活因子，并与RORGT一起诱导IL17A和IL17F基因的表达。因此，T-BET与Runx1的结合将耗尽用于形成Th17细胞中转录活性Runx1/Rorgt复合物的Free Runx1的池。这些数据表明，如果要区分幼稚的CD4+将其分化为Th17细胞，则必须抑制T-bet表达。在发育中的Th17细胞中，T-bet沉默的机制仍然晦涩。最近的两项研究表明，在MicroRNA miR-29b（2011）35：169-181后，TH1细胞中T-BET的表达受到转录后的调节。自然免疫学（2011）12：861-869]。我们使用缺陷型TH17细胞的初步数据表明，T-bet表达在该子集中受到microRNA的负调节。该项目的目的是确定哪个microRNA负责抑制Th17细胞中的T-bet表达。我们在表达T-bet（例如TH0和IL-23处理过的Th17细胞）的TH细胞上进行了微阵列，并在未表达T-bet的Th17细胞中进行了微阵列。差分分析显示，几种microRNA在Th17细胞中高水平表达，并在表达T-bet的TH细胞中减少。此外，生物信息学分析揭示了这些microRNA特有的T-Bet 3？-UTR中存在结合位点。总的来说，这些结果表明，所选的microRNA是Th17分化过程中T-BET表达沉默的良好候选者。为了直接解决这个问题，我们将过度表达Th1细胞中通常具有高水平T-bet的候选microRNA，并研究这些microRNA在Th1细胞中的存在是否对T-bet表达产生负面影响。如果事实证明是这种情况，我们将使这些microRNA在Th17细胞中保持沉默，并研究这种操纵是否会影响IL-17A产生细胞中Th17分化或T-BET的表达。 In addition, we will mutate specific microRNA binding sites in the T-bet 3?-UTR region and determine whether these microRNAs act as direct regulators of T-bet expression, or they act indirectly by affecting signaling pathways that result in T-bet induction (such as IFN-g signaling pathway, for example) The results from this study will reveal the basic understanding of the regulatory mechanisms that act to constrain the T-bet specific pathway that promotes the development of the反对Th1血统。