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Analysis of the transcriptional and post-transcriptional regulation of human iNOS expression

人类 iNOS 表达的转录和转录后调控分析

基本信息

批准号：
273680753
负责人：
Professor Dr. Hartmut Kleinert
金额：
--
依托单位：
Institut für Pharmakologie
依托单位国家：
德国
项目类别：
Research Grants
财政年份：
2015
资助国家：
德国
起止时间：
2014-12-31 至 2018-12-31
项目状态：
已结题

来源：
https://gepris.dfg.de/gepris/projekt/273680753?language=en
关键词：
Analysis transcriptional post regulation human

项目摘要

The high amounts of NO produced by the inducible nitric oxide synthase (iNOS) have beneficial antiviral, antiparasital and antitumoral effects. However, aberrant iNOS induction is involved in the pathophysiology of human autoimmune diseases (rheumatoid arthritis), cancer and septic shock. Therefore it is important to understand the mechanisms by which cells regulate iNOS-related NO production. NO production by iNOS is mainly regulated by regulation of iNOS expression. Beside transcription human iNOS expression is regulated by different post-transcriptional mechanisms. In the 5-untranslated region (5-UTR) upstream of the bona fide iNOS coding sequence (cds) there is a small upstream open reading frame (µORF). Our recent data show that this µORF seems to be translatable and therefore should block the translation of the bona fide iNOS cds. However iNOS protein expression can be observed after induction of cells. Our data indicate that a binding site of the poly-A binding protein (PABP) and an overlapping putative internal ribosomal entry site (IRES) sequence located behind the µORF may be involved in a cap-independent translation of the human iNOS mRNA. In the present application we want to analyze the mechanisms enabling the translation of the iNOS cds in the presence of a µORF. The human iNOS 5-UTR is encoded by exon 1 and in part by exon 2. The stop codon of the µORF is located 50 bp in the front of the intron 1. This hints for a regulation of iNOS expression by the nonsense mediated mRNA decay (NMD). Our data show that siRNA-mediated downregulation of the expression of Upf1 (a central mediator of NMD) enhances human iNOS mRNA- and protein expression. In the present application we want to analyze the involvement of the NMD in human iNOS expression in detail. Korneev et al. describe the expression of a long non coding RNA (lncRNA) with a partial anti-sense homology to the bona fide iNOS gene (as-iNOS-lncRNA) in human cells. Our data show that the expression of the as-iNOS-lncRNA is upregulated by cytokine incubation. In addition down-regulation of as-iNOS-lncRNA significantly reduced cytokine-induced iNOS mRNA- and protein expression. This hints for an regulation of the human iNOS gene by the as-iNOS-lncRNA. In the present application we want to analyze the mechanisms of this regulation. In human C-28/I2 chondrocytes iNOS expression depends on cell differentiation. The mechanisms important for differentiation-dependent iNOS regulation are unknown. Beside changes in the post-transcriptional mechanisms this may be explained by changes in the inducibility of the human iNOS promoter. Our data show, that in contrast to most cells the inducibility of the human iNOS promoter resembles the inducibility of the iNOS mRNA expression (> 100 fold) in differentiated C-28/I2 cells. In the application we want to analyze the differentiation-dependent mechanisms regulating human iNOS expression in C-28/I2 chondrocytes.

由诱导型一氧化氮合酶（iNOS）产生的大量NO具有有益的抗病毒、抗寄生虫和抗肿瘤作用。然而，异常的iNOS诱导参与人类自身免疫性疾病（类风湿性关节炎）、癌症和感染性休克的病理生理学。因此，重要的是要了解细胞调节iNOS相关的NO产生的机制。iNOS产生NO主要通过调节iNOS的表达来调节。除了转录外，人iNOS的表达受不同的转录后机制调节。在真正的iNOS编码序列（cds）上游的5-非翻译区（5-UTR）中，有一个小的上游开放阅读框（µORF）。我们最近的数据表明，这个µORF似乎是可翻译的，因此应该会阻止真正的iNOS cds的翻译。诱导后可观察到iNOS蛋白表达。我们的数据表明，多聚腺苷酸结合蛋白（PABP）的结合位点和位于µORF后面的重叠的假定内部核糖体进入位点（IRES）序列可能参与了人iNOS mRNA的帽非依赖性翻译。在本申请中，我们希望分析在µORF存在下能够翻译iNOS cds的机制。人iNOS 5-UTR由外显子1编码，部分由外显子2编码。µORF的终止密码子位于内含子1前面50 bp处。这暗示通过无义介导的mRNA衰变（NMD）调节iNOS表达。我们的数据表明，siRNA介导的Upf 1（NMD的中央介质）的表达下调增强人iNOS mRNA和蛋白质的表达。在本申请中，我们想要详细分析NMD在人iNOS表达中的参与。Korneev等人描述了在人细胞中与真正的iNOS基因（as-iNOS-lncRNA）具有部分反义同源性的长非编码RNA（lncRNA）的表达。我们的数据表明，as-iNOS-lncRNA的表达上调细胞因子孵育。此外，下调as-iNOS-lncRNA显著降低了马槟榔诱导的iNOS mRNA和蛋白表达。这暗示了as-iNOS-lncRNA对人iNOS基因的调节。在本申请中，我们想要分析这种调节的机制。在人C-28/I2软骨细胞中，iNOS表达依赖于细胞分化。分化依赖性iNOS调节的重要机制尚不清楚。除了在转录后机制的变化，这可能是由在人类iNOS启动子的诱导的变化来解释。我们的数据显示，与大多数细胞相反，人iNOS启动子的诱导类似于分化的C-28/I2细胞中iNOS mRNA表达的诱导（> 100倍）。在本申请中，我们希望分析调节C-28/I2软骨细胞中人iNOS表达的分化依赖性机制。