炎症介质mRNA脱腺苷及稳定性的调控机制研究进展

炎症介质在急性呼吸窘迫综合征等疾病过程中有重要作用。炎症介质mRNA稳定性的调控是RNA调控的一个重要方面。mRNA稳定性调控是一种广泛存在而极为重要的转录后调控方式,mRNA稳定与否直接关系到相应炎症介质表达量的多少。mRNA脱腺苷化是众多mRNA降解起始的开始和限速步骤,直接决定了mRNA稳定性的高低。mRNA脱腺苷并降解的调控途径众多,目前已知的主要有富含AU序列介导、无义介导、微小RNA介导、主要编码区域决定簇介导的途径和mRNA的缓慢脱腺苷途径,在不同细胞、不同环境中占据主导地位的调控途径存在差异。     

碘化钠对人甲状腺细胞IL—6，IFN—γ和TNF—α基因表达的影响

本研究以10^-4M碘化钠（NaI)刺激单层培养的人Graves病（GD）甲状腺滤泡上皮细胞（TEC），采用定性及半定量PCR技术检测刺激前后白介素-6（IL-6）、γ-干扰素（IFN-γ）和肿瘤坏死因子-α（TNF-α）在细胞中的表达水平。结果表明：（1）3例GD组织均含有IL-6及IFN-γmRNA,2例检测到TNF-α基因表达；（2）基础状态下，3例TEC均表达IL-6基础，2例表达TNF-α，而所有样本均无IFN-γmRNA;（3）NaI不能诱导TEC产生IFN-γmRNA，对IL-6mRNA的表达亦无明显影响；（4）1例TNF-α阴性的TEC样本，经NaI刺激后，可表达mRNA，2例原含有TNF-α的TEC经刺激后，其mRNA的表达水平显著增加。提示碘可通过诱导或增强TEC表达TNF-α，导致自身免疫性甲状腺疾病的发生与发展。     

高等真核生物中mRNA稳定性的控制

对于高等真核生物的mRNA来说，尤其是寿命较短的mRNA来说，它的丰度、半衰期及基因表达之间的联系是非常重要的，因为对于这些mRNA来说，它们半衰期的微弱变化会在短时间内使其丰度发生1000倍甚至更大的变化，所以探讨mRNA稳定性控制机理对于研究细胞生长、分化、肿瘤的发生以及抗体对各种应激情况发生的反应有重要意义。     

RNAi及其应用

RNAi（RNA interfeFence，RNAi）指将与内源性mRNA互补的dsRNA（double-strandedRNA，dsRNA）导入细胞后，引起该mRNA特异性的降解，导致mRNA编码的基因不能表达，发生基因沉默。RNAi存在于许多的生物体内，如线虫、果蝇、蛇、斑马鱼、青蛙、老鼠和人等。在自然界中，RNAi有很重要的生物学作用，它既能保护基因对抗由转座子和重复序列引起的不稳定性，也可以对抗病毒等外源性物质的侵入。最近，基因分析证明RNAi在内源基因调控方面也有重要作用。     

P38MAPK、Caspase-8在Fas—AD诱导Bel-7402细胞凋亡中的作用

目的通过P38MAPK抑制剂SB203580及caspase-8抑制剂Ac-IEFD-cho的作用,确定P38MAPK、caspase-8的相互作用关系,进一步揭示Fas—AD诱导Bel-7402细胞凋亡的信号转导机制。方法通过RT—PCR法检测培养的Bel-7402细胞中P38MAPK mRNA、caspase．8 mRNA在Fas．AD、SB203580及Ac—IEFD—cho作用下的表达情况。结果在Fas-AD诱导Bel-7402细胞凋亡中,SB203580和Ac—IEFD—cho能分别抑制p38MAPK mRNA、caspase-8 mRNA的表达。结论在Bel-7402细胞凋亡中,P38MAPK与easpase-8参与Fas—AD凋亡途径,并且在mRNA水平进行相互调节。     

SRSF2基因突变与髓系肿瘤

 剪接因子在真核生物前体mRNA的成熟过程中发挥重要作用,SR蛋白家族是常见的剪接因子之一。 SRSF2属于SR蛋白家族,对mRNA剪接、转录、维持DNA的稳定性及细胞增殖过程中发挥重要作用,SRSF2基因突变在髓系肿瘤（如：骨髓增生异常综合征,慢性粒单核细胞白血病等）检出率较高,并且可能与疾病的表型及预后相关。本文就SRSF基因及其突变的最新研究进展,及其与髓系肿瘤的关系做一综述。     

α—MSH抑制内毒素知症大鼠不同组织表达巨噬细胞移动抑制因子mRNA

目的 研究α－黑素细胞刺激素（α-MSH)对内毒素血症大鼠不同组织表达炎症反应介质巨噬细胞移动抑制因子（MIF）的影响，以探讨α-MSH的抗炎作用机理。方法 SD大鼠腹腔注射LPS建立内毒素血症动物模型，静脉给予α-MSH处理，采用半定量RT－PCR和northern印迹杂交的方法，检测大鼠不同组织MIF mRNA的表达。结果 内毒素血症大鼠垂体、下丘脑、海马、大脑皮层及肾上腺和脾脏MIF mRNA的表达与正常大鼠比较明显增强，α－MSH对大鼠垂体、海马、肾上腺和脾脏MIF mRNA的表达具有明显的抑制作用，但对大脑皮层和下丘脑部位MIF mRNA的表达无明显影响。结论 α－MSH能抑制中枢神经系统和外周器官表达MIF可能在其抗全身炎症反应中起重要作用。     

逆转录病毒介导的反义c—myb对肝星状细胞增殖及I型胶原基因表达的影响

目的：探讨反义c—myb RNA对体外培养的肝星状细胞(HSC)增殖及I型胶原mRNA表达的影响。方法：构建含有反向c—myb基因片段的重组逆转录病毒载体pDOR—myb，将其导入包装细胞PA317中，收获含病毒的培养上清，进一步感染体外培养的大鼠HSC，采用四甲基偶氮唑盐(MTT)法测定细胞增殖反应，采用半定量RT—PCR检测c—myL、α1—I型胶原mRNA表达。结果：成功分离培养大鼠HSC，感染pDOR—myb病毒的HSC自身c—myb表达、细胞增殖及α—I型胶原mRNA表达显著受抑。结论：c—myb在HSC激活增殖过程中起重要作用，反义c—myb基因能抑制HSC增殖及I型胶原基因表达，这提示抑制c—myb表达可能是防治肝纤维化的有效途径。     

bcl—2／bax对不同乳腺癌细胞凋亡的调节作用

目的：了解bcl-2/bax在乳腺癌细胞的p53依赖性凋亡和p53非依赖性凋亡中的作用。方法：用原位杂交的方法检测化疗药物VM-26诱导入乳腺癌细胞株MCF-7和MDA-MB-435S凋亡前后bcl-2和bax的mRNA表达情况。结果：与MDA-MB-435S细胞相比,MCF-7细胞的bcl-2 mRNA水平较高。两株细胞均能在VM-26的作用下发生凋亡。药物诱导后,MCF-7细胞的bcl-2 mRNA表达下降,bax mRNA表达增加;MDA-MB-435S细胞bax mRNA表达增加,bcl-2、mRNA无明显变化。结论：wt p53可在转录水平调节bcl-2/bax的表达,从而介导凋亡作用;bax在p53非依赖性凋亡中也发挥一定作用;bcl-2的高表达可能与肿瘤耐药性有关。     

Mel—la,Mel—lb褪黑素受体在大鼠海马分布的原位杂交研究

Mel-la,Mel-1b为分别从人大脑和视网膜克隆的两种褪黑素膜受体。随着褪黑素的各种重要生理作用的不断发现，对其受体的研究也日益广泛和深入，本文采用原位杂交技术研究了Mel-1a,Mel-1b两种褪黑素膜受体mRNA在海马中的表达和分布，结果表明此两种受体mRNA在成年大鼠海马各区神经元中均有表达。由于褪黑素是通过细胞膜上与G蛋白耦联的Mel-1a,Mel-1b受体而发挥作用的，而海马又是与老化密切相关的脑区，提示褪黑素对海马的老化有调节作用。     

Recruitment and activation of mRNA decay enzymes by two ARE-mediated decay activation domains in the proteins TTP and BRF-1

In human cells, a critical pathway in gene regulation subjects mRNAs with AU-rich elements (AREs) to rapid decay by a poorly understood process. AREs have been shown to directly activate deadenylation, decapping, or 3'-to-5' exonucleolytic decay. We demonstrate that enzymes involved in all three of these mRNA decay processes, as well as 5'-to-3' exonucleolytic decay, associate with the protein tristetraprolin (TTP) and its homolog BRF-1, which bind AREs and activate mRNA decay. TTP and BRF-1 each contain two activation domains that can activate mRNA decay after fusion to a heterologous RNA-binding protein, and inhibit ARE-mediated mRNA decay when overexpressed. Both activation domains employ trans-acting factors to trigger mRNA decay, and the N-terminal activation domain functions as a binding platform for mRNA decay enzymes. Our data suggest that the TTP protein family functions as a molecular link between ARE-containing mRNAs and the mRNA decay machinery by recruitment of mRNA decay enzymes, and help explain how deadenylation, decapping, and exonucleolytic decay can all be independently activated on ARE-containing mRNAs. This describes a potentially regulated step in activation of mRNA decay.     

Unraveling a cytoplasmic role for hnRNP D in the in vivo mRNA destabilization directed by the AU-rich element

AU-rich RNA-destabilizing elements (AREs) have become a paradigm for studying cytoplasmic mRNA turnover in mammalian cells. Though many RNA-binding proteins have been shown to bind to AREs in vitro, trans-acting factors that participate in the in vivo destabilization of cytoplasmic RNA by AREs remains unknown. Experiments were performed to investigate the cellular mechanisms and to identify potential trans-acting factors for ARE-directed mRNA decay. These experiments identified hnRNP D, a heterogeneous nuclear ribonucleoprotein (hnRNP) capable of shuttling between the nucleus and cytoplasm, as an RNA destabilizing protein in vivo in ARE-mediated rapid mRNA decay. Our results show that the ARE destabilizing function is dramatically impeded during hemin-induced erythroid differentiation and not in TPA-induced megakaryocytic differentiation of human erythroleukemic K562 cells. A sequestration of hnRNP D into a hemin-induced protein complex, termed hemin-regulated factor or HRF, correlates well with the loss of ARE-destabilizing function in the cytoplasm. Further experiments show that in hemin-treated cells, ectopic expression of hnRNP D restores the rapid decay directed by the ARE. The extent of destabilizing effect varies among the four isoforms of hnRNP D, with p37 and p42 displaying the most profound effect. These results demonstrate a specific cytoplasmic function for hnRNP D as an RNA-destabilizing protein in ARE-mediated decay pathway. These in vivo findings support an emerging idea that shuttling hnRNP proteins have not only a nuclear but also a cytoplasmic function in mRNA metabolism. The data further imply that shuttling hnRNP proteins define, at least in part, the nuclear history of individual mRNAs and thereby influence their cytoplasmic fate.     

AU-rich elements target small nuclear RNAs as well as mRNAs for rapid degradation

AU-rich elements (AREs, usually containing repeated copies of AUUUA), when present in the 3′-untranslated regions (UTRs) of many mammalian mRNAs, confer instability on their host RNA molecules. The viral small nuclear RNA (snRNA) Herpesvirus saimiri U RNA 1 (HSUR 1) also contains an AUUUA-rich sequence. Here, we report that this ARE induces rapid degradation of HSUR 1 itself and of other snRNAs including HSUR 2 and cellular U1. Mutational analyses of the viral ARE establish that sequence requirements for mRNA and snRNA decay are the same, suggesting a similar mechanism. Moreover, the in vivo degradation activity of mutant AREs correlates with their in vitro binding to the HuR protein, implicated previously as a component of the mRNA degradation machinery. Our results suggest that ARE-mediated instability can be uncoupled from both ongoing translation and deadenylation of the target RNA.     

Efficient replication,and evolution of Sindbis virus genomes with non-canonical 3′A/U-rich elements (NC3ARE) in neonatal mice

Sindbis virus (SIN) is a mosquito-transmitted animal RNA virus. We previously reported that SIN genomes lacking a canonical 19 nt 3′CSE undergo novel repair processes in BHK cells to generate a library of stable atypical SIN genomes with non-canonical 3′A/U-rich elements (NC3AREs) adjacent to the 3′ poly(A) tail [1]. To determine the stability and evolutionary pressures on the SIN genomes with NC3AREs to regain a 3′CSE, five representative SIN isolates and a wild type SIN were tested in newborn mice. The key findings of this study are: (a) all six SIN isolates, including those that have extensive NC3AREs in the 3′NTRs, replicate well and produce high titer viremia in newborn mice; (b) 7–9 successive passages of these isolates in newborn mice produced comparable levels of viremia; (c) while all isolates produced only small-sized plaques during primary infection in animals, both small- and large-sized plaques were generated in all other passages; (d) polymerase stuttering occurs on select 3′ oligo(U) motifs to add more U residues within the NC3AREs; (e) the S3–8 isolate with an internal UAUUU motif in the 3′poly(A) tail maintains this element even after 9 passages in animals; (f) despite differences in 3′NTRs and variable tissue distribution, all SIN isolates appear to produce similar tissue pathology in infected animals. Competition experiments with wt SIN and atypical SIN isolates in BHK cells show dominance of wt SIN. As shown for BHK cells in culture, the 3′CSE of the SIN genome is not required for virus replication and genome stability in live animals. Since the NC3AREs of atypical SIN genomes are not specific to SIN replicases, alternate RNA motifs of alphavirus genome must confer specificity in template selection. These studies fulfill the need to confirm the long-term viability of atypical SIN genomes in newborn mice and offer a basis for exploring the use of atypical SIN genomes in biotechnology.