无义介导的RNA降解及其临床意义

作者:李贺鑫
2021-12-16

作者:李贺鑫 李英 肖飞

【摘要】无义介导的RNA降解(NMD)是真核细胞中mRNA转录产物 质量保证的重要途径。它主要通过外显子连接复合物(EJC)和过长 的3’UTR识别含有提前终止密码子(PTC)的mRNA并进行降解。 NMD也能调控正常的转录产物,参与到诸多正常的生理活动和疾病的 发生发展中。在本综述中,我们讨论了NMD的主要成分,作用机制, 在正常生理过程中的功能,与人类疾病的关系,目前治疗PTC相关疾病 或靶向NMD治疗疾病的一些方法。

【关键字】提前终止密码子( PTC);无义介导的RNA降解;PTC相关 疾病; 治疗

真核细胞具有复杂的基因表达过程,此过程涉及到DNA的转录、加 帽、多腺苷酸化和mRNA的选择性剪接、mRNA的翻译和降解等诸多步骤。 单个碱基突变,框移突变以及选择性剪接等,常产生异常的具有提前终 止密码子(PTC)的mRNA,从而可能翻译成具有潜在毒性的截短蛋白[1- 2]。无义介导的RNA降解(NMD)是机体重要的转录后监控机制,可识别 这种异常的终止密码子,并降解相应的mRNA,避免有害蛋白在细胞内的 积累[3-5]。从低等的单细胞酵母到线虫、植物以及动物,都广泛存在高度 保守的NMD,说明NMD对维持基因组的稳定性和正常表达发挥着不可或缺 的作用[6-8]。除了降解含有突变等产生的具有提前终止密码子的mRNA, NMD对其他一些类型的转录产物也有调控作用,如含有上游开放阅读框 的mRNA(u-ORF)和超长3’非翻译区的mRNA,甚至是非编码RNA[9-10]。 研究表明,NMD调节体内约25%的mRNA的表达水平,参与了细胞的分化、 应激反应、部分遗传性疾病和肿瘤的发生发展等生理病理过程[11-15]。据 估计,约30%的已知疾病相关突变是由于含有PTC的mRNA所致[16],NMD 系统功能的缺失将导致严重的临床表型,如基因敲除NMD中的重要因子 SMG1,SMG6,UPF1或UPF2将直接导致实验小鼠胚胎死亡。本综述概述了NMD的主要成分,NMD的作用的主要 机制,NMD在正常生理过程中的功能,NMD与人类疾病 的关系以及NMD作为潜在的靶点在疾病治疗中的一些探索性应用。

一、 NMD的主要成分 

1. UPF家族:UPF1是人类最重要的NMD因子之 一。UPF1是一个123kDa左右的磷酸蛋白复合物,具有 RNA依赖性ATP酶活性和ATP依赖的RNA解旋酶活性,能 利用水解ATP产生的能量以5’-3’方向水解mRNA[17- 18]。UPF1的N端具有脯氨酸/甘氨酸丰富的区域,该区域 可与SMG1结合,后者使UPF1磷酸化。UPF1的C端富含丝 氨酸和谷氨酰胺(SQ)的结构域,具有14-18个潜在的 磷酸化位点,磷酸化/去磷酸化是调节UPF1功能最重要 的方式[19-20]。研究表明,RNA螺旋酶DHX34的核心区域 与UPF1结合,其CTD区(protruding carboxy-terminal domain)与SMG1结合形成复合物,促进SMG1对UPF1的 磷酸化[21]。UPF1能够有效的重构核糖核蛋白复合物, 一旦被募集到mRNA上并激活,便能扫描整个转录产物 并对核糖核蛋白进行不可逆的重塑,促使mRNA被NMD机 制降解[22]

UPF1对NMD的功能起着至关重要的作用,敲除或沉 默编码UPF1基因将导致含PTC的mRNA稳定性增加。纯合 的UPF1基因敲除小鼠胚胎由于大量诱导细胞凋亡,在 植入后不久即死亡[13]。UPF1不仅是NMD的核心组分之 一,同时也参与了其他的一些生理过程,如参与维持 端粒的稳定[23],促进RNA诱导的沉默复合体与靶标的结 合,诱导的mRNA下调等[24]

2. UPF2:UPF2通常被认为是连接UPF1和UPF3 以引发NMD的支架蛋白[25],鉴于此功能,UPF2具有单 独的结构域分别与UPF1和UPF3结合[26-28]。UPF2的结构 分析显示其具有三个保守的eIF4G(MIF4G)结构域, 前两个结构域主要是用于支撑整个结构,第三个结构 域则介导与UPF3b的结合[29-30]。UPF2 C端的UBD(UPF1 binding domain)与UPF1结合[31]。UPF2可调节UPF1的 活性,UPF1富含半胱氨酸和组氨酸(CH)结构域和SQ 结构域均可抑制UPF1的解旋酶活性,当UPF2与UPF1的 CH结构域结合时,UPF1构象发生改变,这也是UPF1被 SMG1磷酸化的前提[32-33],同时也促进了UPF1的解螺旋 酶活性[25, 34]。UPF2对UPF1的磷酸化不可或缺,在Hela 细胞中,下调UPF2的表达将导致UPF1的磷酸化程度降 低[33]。UPF2在发育和细胞的分化中也起重要作用,在 小鼠胚胎中UPF2的缺失将导致肝脏的终末分化失败, 而成年鼠肝脏中UPF2的缺失导致肝脏脂肪变性和肝脏 内稳态的破坏[35-36]。敲除UPF2的小鼠胚胎在子宫内9.5 天即死亡,敲除造血系统中的UPF2将导致造血干细胞 的完全消失,并很快导致小鼠的死亡[37]

3. UPF3:UPF3是UPF家族中保守性最低的一个[31]。哺乳动物有两个UPF3基因,分别是位于13号染色 体上的UPF3a(也叫UPF3)和位于X染色体上的UPF3b (也叫UPF3X)。而线虫,黑腹果蝇和酿酒酵母都只有 一个UPF3基因[38-40]。 UPF3a和UPF3b都能通过N端的NBD结合域和UPF2结 合[41]。UPF3a和UPF3b主要存在于细胞核中,在核RNA 剪接时被募集到RNA上,并随成熟的mRNA通过核孔转 运到胞质中[26, 28]。虽然UPF3a和UPF3b具有诸多相似的地方,但是对NMD活性影响的强度是不一样的。UPF3b 结合在终止密码子下游时可引发强烈的NMD活性,而 UPF3a在该测定系统中仅能诱发轻微的NMD活性。导致 这种功能差异的原因是,UPF3b的C末端结构域中存在 一段高度保守的序列,但是该序列在UPF3a中则保守性 低[37]。UPF3b通过C末端与EJC复合物的核心成分Y14结 合,在NMD激活过程中起着重要作用[42]。 研究表明,UPF3b在自我更新增加和细胞分化改变 中起着重要的作用[43]。UPF3b敲低导致原代NPC增殖能 力增强但分化能力降低。在原代海马神经元中,UPF3b下调导致神经突生长改变,表明UPF3b在有丝分裂后神 经元中也是必需的[44]。另外一项研究表明,UPF3b错义 突变所导致的神经发育表型是由NMD受损,从而引起的 神经元分化改变引起的[45],因此UPF3b对维持大脑发育 过程中正常的NMD活性是不可缺少的。但是也有研究也 表明,UPF3b对NMD不是必须的,可能存在不依赖UPF3b 的NMD途径。单独敲除UPF3b或联合敲除UPF3a,对NMD 的影响甚小[46]。UPF3b突变而导致其失活的患者中, NMD的活性似乎也影响不大[47]。与UPF1敲除或抑制对 NMD活性起决定性影响不同,小鼠对UPF3b敲除具有 较好的耐受性,能明显的增加含PTC的NMD底物的稳 定性,而且对正常的基因组影响也很小[48]。虽然存在 不依赖UPF3b的NMD的冗余途径,但是在机体发育的 某些阶段,UPF3b对NMD的正常发挥作用是必不可少的。 

4. SMG蛋白:SMG蛋白介导了UPF蛋白的磷酸 化与去磷酸化,包括四个核心成员,SMG1,SMG5, SMG6,SMG7。SMG1是一个410kDa的巨大蛋白,属 于PIKK(phosphatidylinositol 3-kinase-related kinase)家族,介导了UPF1的磷酸化[49]。SMG1的C末 端包含侧翼结构为FRB结构域(FKBP12-雷帕霉素结合 域)的PIKK催化区和两个FAT结构域,形成球状“头 部”区域,而N末端HEAT重复域包含在“臂”区域中[50-51],SMG1磷酸化UPF1的SQ结构域中的丝氨酸残基, 调节NMD的活性[49,52]。SMG激酶缺陷点突变体的过表达 导致PTC依赖性β-珠蛋白mRNA降解显著受到抑制, 而过表达野生型SMG-1则增强NMD对其的降解[49]。 SMG7含有9个反向平行的α螺旋,折叠方式类似 14-3-3蛋白,该14-3-3样结构域包含几个TPR重复 序列,该重复序列介导了蛋白与蛋白之间的相互作 用,具有磷酸丝氨酸残基的保守结合位点。高水平 的保守性表明SMG5和SMG6也含有14-3-3样结构域。 14-3-3样结构域对SMG7的功能很重要,突变SMG7 中14-3-3样结合位点能减弱SMG7与UPF1的结合[53]。 

SMG5,SMG6,SMG7都参与了UPF1的去磷酸化,并 且三者的功能是非冗余的。除了自身具有磷酸酶的 作用,它们还能通过募集其他必须的磷酸酶如PP2A (蛋白磷酸酶2A)促进UPF1的去磷酸化[54-56]。SMG5 和SMG7的14-3-3样结构域在垂直方向相互作用,形 成异二聚体与磷酸化UPF1的S1096和C末端的其他磷酸 化残基相互作用[20,57,58]。与SMG5和SMG7不同,SMG6以 单体的形式发挥功能。SMG5和SMG6的C末端都含有PIN 样区域,其折叠类似RNase H家族核糖核酸酶,是具 有核酸酶活性的磷酸二酯酶[59],SMG6的PIN结构域比 SMG5更加类似RNase H核酸酶。NMD可由PTC附近的核 酸内切酶的剪切诱发,而SMG6就是负责该过程的内切 酶,突变SMG6的PIN结构域中的保守位点,PTC附近的 剪切消失[60-61]。 除了这些已经研究比较透彻的NMD因子,还有 一些其他的因子也参与NMD的过程,并在其中发挥 着重要的作用。如DHX34,促进UPF1和UPF2与EJC的 接触,并促进UPF1的磷酸化[21,62]。其他的因子还有 NBAS[6,62],GNL2,SEL3[63],以及调节NMD过程中mRNA 监控复合物的SMG8,SMG9[64]等。

二、NMD模型

NMD是体内mRNA质量监控最重要的途径之一,其 核心问题是如何区分含PTC的mRNA和正常的mRNA,并 且这种差异如何导致不同的mRNA稳定性。目前较广 为接受的是EJC模型[65-68]和3’UTR模型[69-70]。 1. EJC模型:高等生物中,前体mRNA的剪接在 PTC的识别过程中发挥了重要的作用[70]。剪切募集了 一系列的蛋白,即外显子连接复合体(exon-junction complex,EJC),位于外显子-外显子连接处上游 的20-24个核苷酸处[52]。在哺乳动物中,EJC是以 eIF4A3、Y14、MAGOH、MLN51(RNA-binding protein metastatic lymph node 51 (MLN51))为核心的蛋白 复合体,核心蛋白按照严格的序列排列[71-73];其外周 还具备一系列的其他处于不断变化的蛋白。在正常的 翻译过程中,由于核糖体的滑动,结合在mRNA上的 EJC会被逐个清除[67,74]。EJC-mRNA相互作用严格依赖 于剪接,并且具有位置特异性而非序列特异性。Y14 是一种20kDa核穿梭蛋白,其中心具有RNA结合结构 域(RBD),Y14的RBD和MAGOH在EJC之外彼此紧密相 关,并作为复合物进入细胞核,抑制eIF4A3的解旋酶 活性[75-79]。eIF4A3是eIF4A DEAD-box RNA解旋酶家族的一员,可能作为“RNA夹子”结合在mRNA上[80],而 MLN51可通过SELOR结构域与eIF4A3和RNA结合并促进 eIF4A3的解旋酶活性[77,81]

mRNA在成熟过程中可与多个EJC结合,翻译过程 中这些EJC将会被核糖体移除。核糖体扫描整个mRNA 后,释放因子eRF1和eRF3识别终止密码子,核糖体停 留在终止子处。如果核糖体识别并停留在PTC处,位于 终止密码子下游50-55碱基处的EJC将会被保留。SMG1 及其底物UPF1很快被募集并与eRF1和eRF3形成SURF的 终止后复合体。SURF可与UPF2,UPF3b以及下游的EJC 复合体的的其他蛋白相互作用[82]。SMG1将UPF1磷酸 化,后者以磷酸化特异性的方式募集SMG5,SMG6和 SMG7[20,82]。SMG5-SMG7异二聚体则进一步募集CCR4- NOT脱腺苷酸复合物以及脱帽复合物(DCP complex) 等,促进mRNA的脱帽和脱腺苷酸反应[83-87],从而使 mRNA失去保护,暴露末端,3’RNA片端很快被XRN1 降解,5’RNA被外切体复合物降解[88-89]。另一方面, SMG6具有内切酶的功能,可以剪切PTC附近的mRNA,形 成没有保护的末端促进内切酶对mRNA的剪切[60,90,91],脱 帽反应可以作为正反馈进一步促进SMG6的切割[92]。同 时,UPF1也可与脱帽复合物的DCP1A,DCP2以及PNRC2 作用促进RNA的降解,此过程不依赖于脱腺苷酸化[84,86,93]

2. 异常3’UTR模型:NMD的第二种模型是异常 的3’UTR。该模型认为PTC下游的异常增长的3’UTR 是促进PTC识别的第二种信号。终止核糖体和一组包括 PABPC1(polyA结合蛋白)之间的相互作用是正常情况 下翻译终止和mRNA降解所必须的。因此该模型的关键 在于PTC和3’UTR之间的距离[94]。当正常距离延长时, 正常终止密码子可引发NMD;反之亦然,poly(A)尾 部折叠到PTC附近或通过将PABPC1聚集在PTC附近可以 抑制NMD的激活,并且,研究表明,敲除了PABPC1的 哺乳动物细胞其终止密码子处核糖体的通读能力增加[95],这些都表明PABPC1在促进正确的翻译终止和拮抗 NMD活化方面具有进化上保守的功能[94,96]。 

正常情况下,核糖体遇到终止密码子,eRF1识别 A位点中的终止密码子,其C末端与eRF3形成复合物, 后者N末端与PABPC1的C末端结构域相互作用,促使核 糖体从mRNA上释放,翻译从而终止[97-99]。然而,当PTC 出现时,由于终止的位置提前,翻译终止的下游通常 也不具备正常长度的3’-UTR,因此PABP1不能正确与 eRF3作用,从而核糖体释放过慢[96],没有PABP1与eRF3 作用,UPF1、SMG1等NMD因子能够更直接的与eRF3、 eRF3形成SURF复合物,从而NMD激活,mRNA降解。 正常情况下人类的mRNA的3’-UTR长度在700-800 核苷酸,但是研究却表明,拥有200-300个核苷酸的 3’-UTR长度的mRNA就已经能被NMD影响并部分降解, 而拥有更长3’-UTR的mRNA也并不是都能被NMD识别 或降解,因此虽然过长的3’-UTR是NMD底物的一个 特点,但是3’-UTR的长度并不能很好的预测特定的 mRNA是否是NMD的底物[94,100-102]

那么NMD如何确定不同 长度的3’-UTR的mRNA是否被降解呢?在最近的研究 中, Kishor等证明RNA结合蛋白hnRNP L起着至关重要的 作用。UPF1仅与大多数mRNA瞬时相互作用,但仍然与 NMD靶mRNA结合,因此,UPF1占据提供了mRNA被NMD降 解的可能性。具有高hnRNP L占据率的mRNA倾向于具有 低UPF1占据率,也具有更低的NMD激活可能。hnRNP L 占据率能比3’-UTR的长度更好的反应mRNA是否是NMD 的底物并被降解[103]。 NMD除了能降解含有PTC的mRNA,也能识别并降 解其他的一些mRNA。例如含硒蛋白,上游开放阅读框 (uORF),含有内含子的3’UTR,超长3’UTR,以及 许多非编码RNA[7,104]。UGA在翻译过程中通常被识别为 终止密码子,但是当细胞内硒浓度高时,UGA能够被翻 译为硒代半胱氨酸。如果硒代半胱氨酸掺入最后转录 本最后一个外显子时,能够逃脱NMD的降解,反之, 如果细胞内硒含量低,该UGA又位于外显子连接处的上 游,则会成为NMD的底物被降解[105-107]

三、NMD参与正常的生理过程

1. NMD参与哺乳动物的发育:NMD对哺乳动物正 常发育很重要,敲除某些NMD的成分可对小鼠产生致 死作用。敲除鼠类UPF1同源基因Rent1的实验中,无法 得到纯合子小鼠,说明Rent1对胚胎的存活是必不可 少的。纯合子的Rent1敲除胚胎在植入子宫后很快死 亡,纯合的Rent1敲除囊泡细胞也在简单的细胞增大后 很快凋亡[108]。这些实验结果证明了NMD中最重要的因 子UPF1在胚胎的发育过程中是必须的[109]。UPF1可在一 定程度上使TGFβ的抑制剂SMAD7不稳定,从而促进 TGFβ信号通路。同时UPF1也可以促进编码其他一些 抑制增殖和细胞未分化状态的蛋白的降解。当NMD被 神经表达的微小RNA(miRNA)下调时,神经分化被开 启。这些实验结果证明了NMD中最重要的因子UPF1在 胚胎的发育和细胞的分化过程中是必须的。 敲除小鼠的UPF2基因导致NMD功能丧失肝脏终末 分化失败,出生后很快死亡,而在成年的小鼠中UPF2 的缺失导致肝脏脂肪变性和肝脏内稳态失衡,同时影 响肝脏的再生[35]。在造血系统中UPF2的缺失导致造 血干细胞和祖细胞群快速、完全、持续性的消失。相 反,UPF2缺失小鼠中已经分化的细胞仅轻度受影响, 表明NMD对增殖的细胞更加重要[110]

UPF3b是另一个与细胞的自我更新和分化密切相 关的NMD因子,尤其是脑和神经系统的发育。UPF3b 在神经干细胞和神经元的细胞质和细胞核中都有表 达。错义突变的UPF3b虽然不影响其细胞定位,但是 扰乱了神经元分化并降低了神经突分支的复杂性。在 NMD抑制剂Amlexanox的存在下,神经元分化同样受 到影响[45]。UPF3b依赖性NMD(UPF3b-NMD)在发育过 程中受到多个水平的调节,包括调节UPF3b的表达和 亚细胞定位。神经祖细胞(NPC)中UPF3b-NMD的丧 失导致神经细胞的细胞数量,但是分化受到影响。在 原代海马神经元中,UPF3b-NMD的丧失导致轻微的神 经突生长效应。因此,UPF3b对于神经细胞的分化起 着重要的作用,UPF3b功能的缺失将导致神经细胞的 分化降低[43]。UPF3b中的功能丧失突变导致可变的临 床表现,包括智力残疾(综合征和非综合征),孤独 症,儿童期精神分裂症和注意力缺陷多动障碍[47,111- 113]

SMG蛋白同样也影响胚胎的正常发育。敲除 UPF1,UPF2,UPF3b,或SMG5,SMG7都会对斑马鱼胚 胎的发育,早期分化以及胚胎的存活产生严重的影响[114]。SMG6的完全敲除导致胚胎在囊胚期就死亡,虽然SMG6敲除的胚胎纤维母细胞可以存活,但是失去了 分化为多能干细胞的功能[115]。综上,虽然NMD的不同 因子发挥着不同的作用,但是在胚胎的发育过程中都 起着重要的作用,尤其是干细胞的分化。敲除这些因 子常导致胚胎的死亡或分化失败。值得注意的是,这 些影响可能不仅是影响了NMD的正常功能,也可能同 时影响了这些因子在NMD之外的其他生理功能。 2. 调节细胞的应激反应:当细胞内外的环境改 变,如氧化应激,缺乏能量或氨基酸以及内质网应 激,细胞内的许多基因的表达水平发生变化以适应 环境的改变,初始因子eIF2α被磷酸化,蛋白质的合 成被抑制[116]。而研究表明,eIF2α的磷酸化会抑制 NMD[117],而参与氧化应激稳态维持的一些蛋白如ATF4,ATF-3,CHOP等和维持氨基酸稳态的蛋白如氨基酸合成酶,增加氨基酸通透性的酶则由于NMD受到抑 制而明显增高[7,118,119]

3. 影响病毒的复制:细胞在病毒入侵时,除了 经典的免疫屏障,NMD可能也参与其中,以细胞内作 用的形式限制病毒感染并形成病毒进化,发挥抗病 毒的作用。全基因组siRNA筛选影响Semliki Forest病 毒(SFV)复制的宿主因子(一种正链RNA(+RNA) 病毒),结果显示NMD成分UPF1,SMG5,和SMG7的敲 除导致病毒蛋白和RNA水平升高,释放的病毒滴度更高。NMD组分的消耗导致这些减毒病毒的产生增加超 过20倍。可能的机制是NMD识别并降解某些含有PTC或 者长3’UTR的病毒RNA,这种保护机制在动物和植物 中都存在[120-121]

4. 参与DNA损伤、细胞周期调节:SMG1除了参 与NMD之外,影响DNA修复和未折叠蛋白反应。在植 物中,敲除了SMG1植物对DNA损伤的易感性增加,但 对未折叠的蛋白质诱导剂的耐受性增强。哺乳动物细 胞中也有类似的结果,hSMG-1的消耗导致自发DNA损 伤和对电离辐射(IR)的敏感性增加,敲除SMG-1后单链DNA也增加。SMG-1激酶活性可能在DNA损伤时被 激活,磷酸化特异性DNA修复蛋白,或者NMD失活可 能导致异常mRNA代谢,从而使DNA修复蛋白合成故障[122-124]。UPF1同样也参与了基因组的稳定。UPF1结合 在染色质上,并且随着细胞从G1期到S期逐渐增多。 shRNA介导的UPF1缺失导致人类细胞在S期早期停滞, 诱导ATR依赖性DNA损伤反应[125-126]

四、NMD与疾病及相关的治疗策略

NMD和其活性的高低可以影响疾病的发生和发展。 NMD据估计,约有1/3的遗传疾病是由点突变或者移码 突变导致的PTC所致[15,127]。NMD是一把双刃剑,一方面 可以通过降解截短的有害蛋白,防止其积累对细胞产 生毒性;另一方面,有些疾病中截短的蛋白也具有部 分的活性,可以发挥部分的生理功能,NMD对其的降解 会加重疾病的症状。

NMD缓解疾病的一个例子是β-地中海贫血。出生 后人类的血红蛋白是由α-亚基和两个β-亚基组成的 异四聚体。大多数隐性β-地中海贫血患者由β-珠蛋 白基因的第一个或第二个外显子中的无义突变引起, 含有PTC的mRNA被NMD降解。在杂合子中,未受影响的 等位基因仍能够产生正常的β-珠蛋白,因此基本表现 为正常。但是在一些罕见的NMD不敏感的患者中,截短 的β-珠蛋白前体产生过多,对正常的β-珠蛋白产生 显性负性效应,因此这些患者的症状比较严重[128-129]。 NMD保护了绝大部分的杂合子的β-地中海贫血的患者 免于临床症状的贫血。

对于另外的一些疾病,NMD降解截短的蛋白则会加 重病人的临床症状,如囊性纤维病,营养不良以及多 囊肾等[15,130]。杜氏肌营养不良的患者由于基因突变导 致了无义突变,该突变形成的PTC可被NMD识别并降解 具有正常蛋白部分甚至全部功能的C端截短的抗肌营养 不良蛋白。由于NMD的降解,导致了单倍剂量不足,因 而患者具有严重的临床症状。而部分C端截短蛋白逃脱 了NMD降解的患者则具有比较轻的临床症状[131-132]。 1. NMD与肿瘤:NMD与肿瘤之间也密切相关,可 以通过调节原癌基因、抑癌基因的正常表达来影响癌 症的发生发展。UPF1在肺鳞癌,肝细胞癌,胃癌等许 多肿瘤中被报道由于启动子区的高度甲基化而表达下调,NMD活性降低,并且低UPF1表达的病人往往具有 更差的预后[133-136]。在胃癌细胞中过表达UPF1可以抑制 细胞的增殖、迁移、侵袭和细胞周期,促进细胞的凋 亡,这种抑制作用可被过表达MALAT1逆转[135]。UPF1可 以抑制肺腺癌(ADC)细胞和肝癌细胞的上皮间充质转 化(EMT)[133,137],肝癌细胞中UPF1可以通过抑制ABCC2 的表达抑制EMT并促进对化疗的敏感性[137]。UPF1的降 低导致了NMD的降低,Sma7表达上调,TGF-β信号通 路活性增强,从而促进了肿瘤的发展,UPF1可望作为 这些癌症的一个治疗靶点[133-134]。 肿瘤中UPF1除了表达下调,还可以发生无功能的 突变。胰腺腺鳞癌(ASC)是一种知之甚少但侵袭和转 移能力强,预后极差的癌症,目前没有合适的分子诊 断指标。研究发现,在ASC中,UPF1常发生突变导致错 误剪接,NMD活性降低,NMD底物积累。UPF1突变是目 前已经报道的唯一一种ASC分子指标[138]。炎性肌纤维 母细胞瘤中也有报道UPF1的突变,NMD对底物的降解减 少,从而NIK-依赖的NF-κB途径活性增高,促进免疫 细胞的浸润[139]

2. 靶向NMD的治疗手段:(1)反义寡核苷酸: 反义寡核苷酸(ASOs)通过Watson-Crick碱基配对特 异性结合其RNA靶标以形成DNA-RNA杂合双链,其中 的RNA链很快被核酸内切酶RNase H1降解,从而使相 应的mRNA失活[136]。在小鼠模型中,针对UPF3b的ASO (UPF3b-ASO)治疗能够降低NMD的活性,明显的增加 含PTC的抗肌萎缩蛋白mRNA和凝血因子IX mRNA,并且 对正常的基因组影响很小。同时连用促进翻译通读的 药物将进一步增强这种效果,甚至可以产生有功能的 凝血因子IX[140]。体外实验中,针对外显子2Q39X无义突 变(HBB-T39)的基因,ASO联合促进通读药物G418能 够有效的恢复全长蛋白的表达[141]。ASO也可以靶向由 于剪接或者突变产生PTC的异常mRNA,在β-地中海贫 血和抗肌营养不良蛋白的mRNA中都有过试验,并且效 果不错[142-143]。(2)氨基糖苷类药物:氨基糖苷类能 够结合核糖体的解码中心并降低密码子-反密码子配对 的准确性,抑制终止密码子的识别,增加终止密码子 的通读性。动物模型和人体中都用氨基糖苷类药物庆 大霉素治疗囊性纤维病和杜氏肌营养不良,结果显示 氨基糖苷类药物可以促进终止密码子的通读,增加蛋 白的全长表达[144-146]。氨基糖苷类药物G418也常与其 他的药物联用,具有更好的效果[141,147]。但是氨基糖 苷类药物的肾毒性和耳毒性较大,一定程度上限制了 它的应用。(3)NMD抑制剂:化疗药物多柔比星联 合小分子的NMD抑制剂NMDI-1增强进化疗效果,促进 细胞的死亡[148]。在含PTC的突变型p53细胞中,小分 子NMD抑制剂NMDI-14联合促进翻译通读药物G418 能 够破坏SMG7-UPF1的相互作用,抑制NMD,恢复全长 p53的表达,上调p53下游转录物的水平,促进细胞的 死亡,并且对于p53野生型细胞的活性影响不大[147]。 Nickless等高通量筛选了一千多种小分子药物,发现 强心苷类的Na-K-ATPase抑制剂能够提高细胞内钙离 子浓度,有效的抑制NMD[149]。另外,非特异性的SMG1 抑制剂,咖啡因、渥曼青霉素等也能抑制NMD的活 性,但是同时也抑制了PI3K家族成员的活性,对细胞 的其他代谢和生理功能影响较大[52,150]。(4)通过特 异的siRNA抑制NMD:肿瘤不受免疫系统控制的主要原 因是肿瘤细胞不表达有效的肿瘤排斥抗原(TRAs)。 在皮下和转移性肿瘤模型中,与寡核苷酸适体配结合 的靶向NMD因子的特异性siRNA可诱导肿瘤细胞产生新 的潜在的抗原决定簇和免疫介导的排斥反应,明显抑 制肿瘤细胞的生长。并且适配体-siRNA的免疫原性风 险也很低,可望作为临床治疗肿瘤的潜在方法之一[134]

五、 结论 

NMD是转录后水平保证基因组稳定性的重要机 制,它主要通过降解含PTC的mRNA从而减少C端截短 的蛋白的积累来发挥作用。NMD的功能涉及到多个方 面,包括发育和分化,应激反应,参与DNA的损伤修 复,部分遗传病的发生,肿瘤的发生发展等多个方 面。靶向NMD也是目前肿瘤和某些遗传病治疗的热 点,然而目前还局限于体外实验和动物实验,不曾在 临床应用于人类疾病的治疗。但是NMD是把双刃剑, 抑制或激活都可能在治疗疾病的同时对正常的组织细 胞产生副作用。因此,如何提高靶向NMD治疗的特异 性,权衡NMD活性强弱,最终应用到临床疾病的治疗 是未来研究需要解决的问题。 

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