RNA干扰技术新进展

20世纪90年代初,科学家在矮牵牛花中导入与花青素合成有关的查尔酮合成酶基因,原以为转基冈的牵牛花会变得更鲜艳,结果却发现一些花反而变成白色,这种现象引起人们的高度关注,之后的研究将这种过度表达内源基因而引发的基因沉默称为共阻遏.

We hypothesized that RNA interference to silence Nogo-66 receptor gene expression in bone marrow mesenchymal stem cells before transplantation might further improve neurological function in rats with spinal cord transection injury. After 2 weeks, the number of neurons and BrdU-positive cells in the Nogo-66 receptor gene silencing group was higher than in the bone marrow mesenchymal stem cell group, and significantly greater compared with the model group. After 4 weeks, behavioral performance was signiifcantly enhanced in the model group. Af-ter 8 weeks, the number of horseradish peroxidase-labeled nerve ifbers was higher in the Nogo-66 receptor gene silencing group than in the bone marrow mesenchymal stem cell group, and signiifcantly higher than in the model group. The newly formed nerve ifbers and myelinated ner ve ifbers were detectable in the central transverse plane section in the bone marrow mesenchymal stem cell group and in the Nogo-66 receptor gene silencing group.

To stop the progression of Alzheimer’s disease in the early stage, it is necessary to identify new therapeutic targets. We examined striatal-enriched phosphatase 61 expression in the brain tissues of 12-month-old APPswe/PSEN1dE9 transgenic mice. Immunohistochemistry showed that al-enriched phosphatase 61 protein expression was significantly increased but phosphorylated N-methyl-D-aspartate receptor 2B levels were significantly decreased in the cortex and hippocam-pus of APPswe/PSEN1dE9 transgenic mice. Western blotting of a cel model of Alzheimer’s disease consisting of amyloid-beta peptide (1-42)-treated C57BL/6 mouse cortical neurons in vitro showed that valeric acid (AP5), an N-methyl-D-aspartate receptor antagonist, significantly inhibited amyloid-beta 1-42-induced increased activity of striatal-enriched phosphatase 61. In addition, the phos-phorylation of N-methyl-D-aspartate receptor 2B at Tyr1472 was impaired in amyloid-beta 1-42-treated cortical neurons, but knockdown of striatal-enriched phosphatase 61 enhanced the phosphorylation of N-methyl-D-aspartate receptor 2B. Col ectively, these findings indicate that striatal-enriched phosphatase 61 can disturb N-methyl-D-aspartate receptor transport and inhibit the progression of learning and study disturbances induced by Alzheimer’s disease. Thus, al-enriched phosphatase 61 may represent a new target for inhibiting the progression of Alzheimer’s disease.

Tol-like receptor 3 protein expression has been shown to be upregulated during cerebral ische-mia/reperfusion injury in rats. In this study, rat primary cortical neurons were subjected to oxy-gen-glucose deprivation to simulate cerebral ischemia/reperfusion injury. Chemical y synthesized smal interfering RNA (siRNA)-1280,-1724 and-418 specific to tol-like receptor 3 were transfected into oxygen-glucose deprived cortical neurons to suppress the upregulation of tol-like receptor 3 protein expression. Western blotting demonstrated that after transfection with siRNA, tol-like ceptor 3 protein expression reduced, especial y in the tol-like receptor 3-1724 group. These results suggested that siRNA-1724 is an optimal sequence for inhibiting tol-like receptor 3 expression in cortical neurons fol owing oxygen-glucose deprivation.

Inhibition of neurite growth, which is mediated by the Nogo-66 receptor (NgR), affects nerve regeneration following neural stem cell (NSC) transplantation. The present study utilized RNA interference to silence NgR gene expression in NSCs, which were subsequently transplanted into rats with traumatic brain injury. Following transplantation of NSCs transfected with small interfering RNA,typical neural cell-like morphology was detected in injured brain tissues, and was accompanied by absence of brain tissue cavity, increased growth-associated protein 43 mRNA and protein expression,and improved neurological function compared with NSC transplantation alone. Results demonstrated that NSC transplantation with silenced NgR gene promoted functional recovery following brain injury.

RNA干扰技术的临床应用需要更多的安全性研究

看到关于RNA干扰(RNAi)技术的一期和二期临床试验的文章发表,是一件令人兴奋的事.RNA干扰是一种可以选择性地(序列特异性地)使目标基因沉默的技术.已经发表的临床试验文章是关于将该技术应用于治疗人类的少数几种疾病的,虽然有人相信该技术有被用于成千上万种疾病的潜力.对于4种潜在RNAi产品进行的6项临床试验的文章的发表,象征着这种迷人的基因沉默技术已经距临床应用很近了,从而很快将给临床医师提供与诸如恶性肿瘤、病毒感染、某些年龄相关性变性性疾病,以及可能许许多多与基因的突变或过度表达相关疾病的战斗中可用的崭新武器.本篇评论旨在根据国际医学期刊上发表的文献了解RNAi技术安全性研究方面的概况,包括在临床和动物实验研究中对安全性问题的研究情况,并对将此技术安全应用于人类作出一些建议.

针对ATM基因RNA干扰质粒的构建及鉴定

目的:构建针对ATM基因的RNA干扰表达质粒pRiATM,并观察其抑制SPCA1人肺癌细胞ATM表达的效果.方法:构建针对ATM基因的短发夹状小干扰RNA真核表达载体pRiATM,并短暂转染SPCA1细胞,采用荧光定量RT-PCR和Western blotting观察其抑制SPCA1细胞ATM表达的效果.结果:pRiATM1和pRiATM2转染SPCA1细胞后,与转染pRiGFP非特异性对照组相比,ATM mRNA水平分别下降86.4%和77.6%(两组均P＜0.01).与转染pRiGFP对照组相比,pRiATM1组和pRiATM2组ATM蛋白表达水平分别是对照组的4.3%和10.6%(均P＜0.01),以pRiATM1抑制ATM表达效果显著.结论: pRiATM质粒构建成功,转染SPCA1细胞后可以明显抑制ATM基因的表达.

RNA干扰及其在癌症治疗中的应用

RNA interference(RNAi), a highly conserved process of post-transcriptional gene silencing, can be induced by small interfering double-stranded RNA that mediate sequence-specific mRNA degradation. In the past several years, RNAi has been widely used as both an experimental tool to study mammalian gene function and a potential therapeutic approach to treat human diseases. In addition, some new proteins which involve in RNAi pathway have been characterized in mammalian cells. Here, we summarize the various molecules in RNAi, mechanism of action, and the current therapeutic applications in cancers.

应用基因敲除和RNA干扰技术研究睾丸特异性新基因

睾丸特异性基因(testis-specific-gene),是指主要在睾丸组织中表达,面在机体其他组织中不表达或者极低表达的基因.睾丸特异性基因主要与睾丸功能有关,此类基因的正常表达多与睾丸雄激素产生、精子发生有关.

siRNA阻断NF-κB信号通路联合5-Fu对食管鳞癌细胞周期的影响

目的 利用RNAi技术特异性的抑制NF-Κb亚单位p65的表达,探讨在食管鳞癌细胞中将NF-Κb作为基因治疗靶点的价值.方法 将荧光素标记的对照siRNA转染到食管鳞癌细胞中观察转染效率,p65 siRNA转染到EC9706和Eca109细胞中,使用Western blot的方法检测p65和Cyclin D1蛋白的表达,使用EMSA法检测转染前后p65与DNA结合活性的变化;流式细胞仪检测p65 siRNA与5-Fu(327 μg/ml)单独或联合应用,对食管鳞癌细胞周期的影响.结果 荧光素标记的siRNA转染食管鳞癌细胞的效率可达90%以上.在转染后的EC9706和Eca109细胞中,p65和CyclinD1蛋白的表达水平下调;NF-Κb与DNA的结合活性明显下降;G0/G1期的细胞开始增加,同时S期的细胞逐渐减少;当转染p65 siRNA细胞联合使用5-Fu时,G0/G1期的细胞明显增加,同时S期的细胞明显减少.结论 p65siRNA可以特异性的阻断NF-Κb信号通路,下调NF-Κb下游基因中细胞周期蛋白CyclinD1的表达,表明活化的NF-Κb信号通路可以成为食管鳞癌基因治疗中一个重要的分子靶点.

AIM:To investigate the regulation mechanism for insufficient KChIP 2 expression induces Ito,f downregulation and arrhythmogene-sis in cardiac hypertrophy .METHODS:Bidirectional manipulations of MG 53 expression were performed by adenoviral overexpression of MG53 or knockdown of MG53 with RNA interference in neonatal rat ventricular myocytes with or without PE stimulation .Ito,f was re-corded with patch clamp in whole-cell mode 48 h after adenoviral transfection .Then the WT or MG53 knockout ( MG53 -/-) mouse model of left ventricular hypertrophy induced by transverse aortic constriction ( TAC) were used to detect the susceptibility to ventricu-lar arrhythmia.RESULTS: Here, we show muscle-specific MG53 regulates KChIP2 expression and Ito,f densities, where they are downregulated in hearts from MG53 knockout mice and MG53 knockdown rat cardiomyocytes , but upregulated in MG53 overexpressed cells.MG53 expression is decreased in phenylephrine ( PE)-induced cardiomyocyte hypertrophy and restoration of MG 53 rescues PE-induced downregulation of KChIP2 and Ito,f.Furthermore, MG53 is decreased in a mouse model of hypertrophy induced by transverse aortic constriction and ablation of MG 53 increases the susceptibility to ventricular arrhythmia by exaggerating Ito,f remodeling.CON-CLUSION:These findings establish MG53 as a novel regulator of Ito,f and its central role in arrhythmogenesis in hypertrophy .

AIM:Increasing evidence suggests that carbohydrate-binding proteins play an essential role in tumor growth and metastasis .Ga-lectin-3, a multifunctional protein of an expanding family of β-galactoside-binding animal lectins , is the major nonintegrin cellular laminin-binding protein , and is implicated in a variety of biologic events , such as inflammation and angiogenesis .Because galectin-3 expression was shown to participate in mediating tumor angiogenesis and initiate signaling cascades in several diseases .We hypothe-sized that galectin-3 may promote pulmonary vascular endothelial neovascularization .METHODS:Hypoxic and MCT rat model of pul-monary artery remodeling was used .The mRNA and protein levels of galectin-3 in rats were measured by in situ hybrization and West-ern blot analysis.Endothelial cell (EC) proliferation, migration and tube formation were measured using MTT , cell scratch and Matri-gel assays, respectively.Protein expression was quantitated by Western blot analysis .LC 3A/B staining was detected with cellular im-munofluorescence staining .RESULTS:We found that galectin-3 was localized on the intima and adventitial wall .Galectin-3 was in-creased after rat hypoxia and MCT administration .Galectin-3 promoted EC proliferation , migration and tube formation , while its roles were reversed by RNA interference.Galectin-3 induced Atg 5, Beclin-1, LAMP-2, and LC 3A/B expression increases.Galectin-3 al-so increased LC 3A/B staining in ECs.Akt/mTOR and GSK-3βsignaling pathways were activated after galectin-3 treated ECs using its specific phosphorylation antibodies , while blocked it with LY294002 inhibited cell autophagy and EC dynamic alterations induced by galectin-3.CONCLUSION:These findings demonstrate that galectin-3 can induce an Akt signaling cascade leading to cell autoph-agy, and then the differentiation and angiogenesis of pulmonary artery endothelial cells .

FAK的活化促进悬浮肺肿瘤细胞的积聚、凋亡抑制与增殖

[目的]研究在悬浮状态下细胞聚集与细胞凋亡和增殖的关系,以及参与细胞聚集的分子信号传导.[方法]镜下观察不同肺上皮细胞悬浮状态聚集作用的改变;DNA琼脂糖凝胶电泳检测细胞凋亡;用MTT和台盼蓝细胞排斥实验检测细胞增殖;磷酸化蛋白的检测用免疫沉淀和Western blot;RNAinterference沉默实验采用逆转录病毒载体感染的稳定表达细胞株.[结果]肿瘤细胞在polyHEMA悬浮状态下脱离细胞外基质不仅能够存活,而且不同细胞形成大小不同的聚集体:人类大细胞肺癌H460和人肺腺癌H1792细胞株的细胞聚集体大而紧密;人肺腺癌A549和SK-LU-1细胞聚集体小而松散;正常狗肾上皮细胞MDCK几乎不形成聚集体,这与它们的恶性程度来源有关.在悬浮状态下正常细胞MDCK发生凋亡.细胞聚集体间的相互作用能够促进悬浮状态下肿瘤细胞的增殖.磷酸化FAK的水平和聚集体的大小有关,沉默FAK蛋白的表达能够部分地阻断肿瘤细胞聚集体的形成.[结论]肿瘤细胞脱离细胞外基质形成的聚集能力是判断肿瘤恶性程度和转移特性潜在的标志物.聚集体细胞间的相互作用能够抑制细胞凋亡和促进细胞增殖.聚集体的形成与FAK磷酸化有关.

miRNA146a对软骨细胞VEGF表达的影响

目的：研究在正常软骨细胞及中期和晚期骨关节炎（osteoarthritis，OA）软骨细胞中激活或抑制 miRNA146a后血管内皮生长因子（vascular endothelial groxth factor，VEGF）表达的变化规律，探索 miRNA146a 在 OA 发生、发展中的作用。方法收集正常人膝关节软骨4例，OA 患者膝关节软骨12例。OA 组又依据 Kellgren-Lawrence 的放射学诊断标准分为中期和晚期 OA。通过化学转染的方法转染 miRNA146a mimic 或 miRNA146a inhibitor 于各组软骨细胞，测定激活或抑制 miRNA146a 后，VEGF 蛋白水平表达的变化。结果正常人软骨细胞中激活 miRNA146a 后，VEGF 的蛋白水平较对照组及抑制组均出现明显的上升，结果具有统计学意义（ P <0.05）；抑制 miRNA146a 后，VEGF 蛋白水平下降，结果没有统计学意义（P≥0.05）。中期 OA 软骨细胞表达 VEGF 水平高于晚期 OA 软骨细胞，差异具有统计学意义（P <0.05）。中期 OA 软骨细胞激活 miRNA146a 后，VEGF 的蛋白水平上升，结果具有统计学意义（P <0.05）；抑制 miRNA146a 后，VEGF 的蛋白水平下降，结果具有统计学意义（P <0.05）。晚期 OA 软骨细胞激活 miRNA146a 后， VEGF 的蛋白水平上升，结果具有统计学意义（P <0.05）；晚期 OA 软骨细胞抑制 miRNA146a 后，VEGF 的蛋白水平下降，结果不具有统计学意义（P≥0.05）。结论 miRNA146a 可调控软骨细胞 VEGF 表达，从而参与 OA 的发生、发展。关键词：骨关节炎；miRNA146a；血管内皮生长因子；RNA 干扰

采用RNA干扰技术抑制A549细胞表皮生长因子受体表达

AIM: To investigate the biological features of A549 cells in which epidermal growth factor (EGF) receptors expression were suppressed by RNA interference (RNAi). METHODS: A549 cells were transfected using short small interfering RNAs (siRNAs) formulated with Lipofectamine 2000. The EGF receptor numbers were determined by Western blotting and flowcytometry. The antiproliferative effects of sequence specific double stranded RNA(dsRNA) were assessed using cell count, colony assay and scratch assay. The chemosensitivity of transfected cells to cisplatin was measured by MTT. RESULTS: Sequence specific dsRNA-EGFR down-regulated EGF receptor expression dramatically. Compared with the control group, dsRNA-EGFR reduced the cell number by 85.0 %,decreased the colonies by 63.3 %, inhibited the migration by 87.2 %, and increased the sensitivity of A549 to cisplatin by four-fold. CONCLUSION: Sequence specific dsRNA-EGFR were capable of suppressing EGF receptor expression, hence significantly inhibiting cellular proliferation and motility, and enhancing chemosensitivity of A549 cells to cisplatin. The successful application of dsRNA-EGFR for inhibition of proliferation in EGF receptor overexpressing cells can help extend the list of available therapeutic modalities in the treatment of non-small-cell lung carcinoma (NSCLC).