siRNA对肝纤维化的抑制作用

目的：研究肝星状细胞（use）中smad2特异性小干扰RNA（siRNA）对I型胶原表达的抑制作用,探讨抗肝纤维化的基因治疗新方法。方法：设计合成靶向Smad2基因的siRNA,将筛选成功的siRNA瞬时转染入体外培养的肝星状细胞（HSC）,并给予转化生长因子p（TGF．B）刺激,应用RT—PCR和Westernblot技术检测对照组与实验组I型胶原mRNA水平和蛋白水平表达差异,研究siRNA对I型胶原表达的抑制作用。结果：siRNA能明显降低肝星状细胞中Smad2的RNA和蛋白的表达水平,证实筛选的siRNA有效,能特异性抑制Smad2的基因表达;TGF-β刺激肝星状细胞后,与对照组比较,siRNA转染组细胞外基质（ECM）成分I型胶原的表达水平明显降低（P〈0．05）。结论：siRNA能够抑制TGFβ对肝星状细胞的激活,阻断TGFB—Smads传导通路,使I型胶原分泌下调,有效抑制TGFB诱导的肝纤维化。     

Lentiviral Vector-Mediated siRNA Knockdown of c-MYC: Cell Growth Inhibition and Cell Cycle Arrest at G2/M Phase in Jijoye Cells

Inhibition of c-MYC has been considered as a potential therapy for lymphoma treatment. We explored a lentiviral vector-mediated small interfering RNA (siRNA) expression vector to stably reduce c-MYC expression in B cell line Jijoye cells and investigated the effects of c-MYC downregulation on cell growth, cell cycle, and apoptosis in vitro. The expression of c-MYC mRNA and protein levels were inhibited significantly by c-MYC siRNA. The c-MYC downregulation resulted in the inhibition of cell proliferation and cell cycle arrest at G2/M phase, which was associated with decreased expression of cyclin B and cyclin-dependent kinase 1 (CDK1) and increased expression of CDK inhibitor p21 proteins. In addition, downregulation of c-MYC induced cell apoptosis characterized by DNA fragmentation and caspase-3 activation. Taken together, these results suggest that lentiviral vector-mediated siRNA for c-MYC may be a promising approach for targeting c-MYC in the treatment of Burkitt lymphoma.     

Gene specific siRNA selector

Small interfering RNA (siRNA) is used in functional genomics applications to decrease the expression of a target gene, which may yield a biological effect that suggests a function for the target gene. The siRNA design tool scans a target gene for candidate siRNA sequences that satisfy user-adjustable rules. Selected candidates are then screened to identify those siRNA sequences that are specific to the gene of interest.     

Combination of active and inactive siRNA targeting the mitotic kinesin Eg5 impairs silencing efficiency in several cancer cell lines

Gene silencing by RNA interference (RNAi) has proven to be a powerful tool for investigating gene function in mammalian cells. Combination of several short interfering RNA (siRNA) targeting the same gene is commonly used to improve RNA interference. However, in contrary to the well-described mechanism of RNAi, efficiency of single siRNA compared to pool remains poorly documented. We addressed this issue using several active and inactive siRNA targeting Eg5, a kinesin-related motor involved in mitotic spindle assembly. These siRNA, used alone or in combination, were tested for their silencing efficiency in several cancer cell lines. Here we show that presence of inactive Eg5 siRNA in a pool dramatically decreases knockdown efficacy in a cell line- and dose-dependent manner. Lack of inhibition by unrelated siRNA suggests that a competition may occur during siRNA incorporation into RNA-induced silencing complexes (RISCs) along with the target mRNA. Altogether, our results, which need to be confirmed with additional inactive siRNA, indicate that combination of siRNA may not increase but instead decrease silencing efficiency.     

siRNA表达载体的构建及其表达

本研究用限制性内切酶消化质粒pCMV-tag-2B,除去巨细胞病毒(Cytomegalovirus,CMV)启动子核苷酸序列,剩下的核苷酸序列作为构建表达siRNA(Small interfering RNA,siRNA)载体的前体。依据文献提供的扩增H1RNA启动子核苷酸序列的引物序列合成一对引物,以带有H1RNA启动子序列的质粒DNA为模板扩增HIRNA启动子序列,插入前体,构建SiRNA的表达载体pCH1。另外将H1RNA启动子插入pGEM．1lfz相应位点,构建瞬时表达载体pGHl。依据EGFP的有效SiRNA抑制位点,合成两条分别为64bp的核苷酸链,通过体外退火,形成双链,然后插入已构建的两个表达载体。将这两个载体分别与表达EGFP蛋白的质粒pEGFP．N3共转染Bel．7402细胞,观察siRNA对EGFP的抑制效应。研究结果表明构建的载体有效表达了siRNA,这些载体可以用于与siRNA相关抗病毒治疗性试验研究。     

Stable inhibition of hepatitis B virus expression and replication by expressed siRNA

RNA interference might be an efficient antiviral therapy for some obstinate illness. Here, we studied the effects of hepatitis B virus (HBV)-specific 21-nt small interfering RNAs (siRNA) on HBV gene expression and replication in 2.2.15 cells. Seven vectors expressing specific hairpin siRNA driven by the RNA polymerase II-promoter were constructed and transfected into 2.2.15 cells. In the cell strain that can stably express functional siRNA, the HBV surface antigen (HBsAg) and the HBV e antigen (HBeAg) secretion into culture media was inhibited by 86% and 91%, respectively, as shown by an enzyme-linked immunosorbent assay. Immunofluorescence and Western blot indicated similar results. HBV DNA was markedly restrained by 3.28-fold, as assessed by the fluorescent quantitation PCR. Moreover, the HBV mRNA was significantly reduced by 80% based on semiquantitative RT-PCR. In conclusion, the specific siRNA can knock down the HBV gene expression and replication in vitro, and the silence effects have no relationship with interferon response.     

Antibody-directed cell-type-specific delivery of siRNA

Over the past four years, chemically synthesized short interfering RNA (siRNA) has become the standard tool for specific silencing of gene expression in vitro. The most difficult task in transferring this technology to an in vivo setting is to develop appropriate delivery strategies. With this aim, Song et al. recently reported the development of antibody-protamine fusion proteins as vehicles for receptor-directed delivery of siRNA. When a mixture of siRNA targeting tumor-related genes was administered in this way, tumor growth was inhibited in an engineered melanoma model, demonstrating the therapeutic potential of this technology. However, several challenges remain to be overcome before targeted gene silencing can become a reality for patients.     

抑制VEGF基因表达对乳腺癌细胞细胞周期的影响

目的：研究针对VEGF基因的siRNA（small interferenceRNA）对乳腺癌MCF-7细胞细胞周期的影响。方法：依据Promega公司在网上提供的设计软件,设计针对VEGF基因的siRNA,合成DNA模板,体外转录合成siRNA。脂质体转染法将合成的siRNA转染入MCF-7细胞,以未转染细胞以及错义序列siRNAscr转染细胞为对照。用细胞计数法检测siRNA对MCF-7细胞增殖的影响：流式细胞法检测细胞周期变化,RT—PCR法比较转染前后p21、CyclinDl表达水平的变化,Westemblot检测转染前后磷酸化ERK的表达。结果：细胞计数法结果显示,转染24h后siRNA明显抑制MCF-7细胞增殖,转染48h后,抑制效率稳定。siRNA转染后能有效地抑制MCF-7细胞的增殖,阻滞细胞周期于G0／G1期,S期细胞明显减少,G0／G1期细胞比例逐渐增多;p21mRNA表达显著上调,抑制CyclinD1mRNA及磷酸化ERK蛋白的表达。结论：体外转录合成的siRNA可能通过上调细胞周期蚤白激酶抑制剂p21的表达,下调CyclinDl及磷酸化ERK的表达,将细胞周期阻滞于G0／G1期,从而显著抑制MCF-7细胞的增殖。     

Insights into effective RNAi gained from large-scale siRNA validation screening

Transfection of chemically synthesized short interfering RNAs (siRNAs) enables a high level of sequence-specific gene silencing. Although siRNA design algorithms have been improved in recent years, it is still necessary to prove the functionality of a given siRNA experimentally. We have functionally tested several thousand siRNAs for target genes from various gene families including kinases, phosphatases, and cancer-related genes (e.g., genes involved in apoptosis and the cell cycle). Some targets were difficult to silence above a threshold of 70% knockdown. By working with one design algorithm and a standardized validation procedure, we discovered that the level of silencing achieved was not exclusively dependent on the siRNA sequences. Here we present data showing that neither the gene expression level nor the cellular environment has a direct impact on the knockdown which can be achieved for a given target. Modifications of the experimental setting have been investigated with the aim of improving knockdown efficiencies for siRNA-target combinations that show only moderate knockdown. Use of higher siRNA concentrations did not change the overall performance of the siRNA-target combinations analyzed. Optimal knockdown at the mRNA level was usually reached 48-72 hours after transfection. Target gene-specific characteristics such as the accessibility of the corresponding target sequences to the RNAi machinery appear to have a significant influence on the knockdown observed, making certain targets easy or difficult to knock down using siRNA.     

siRNA抑制c—myc基因的表达对宫颈癌细胞增殖的影响

目的：利用siRNA（small interference RNA）技术研究C-myc基因的对宫颈癌HeLa细胞增殖的影响。方法：依据Promega公司在网上提供的设计软件,设计针对C-myc基因的siRNA,合成DNA模板,体外转录合成siRNA。通过阳离子聚合物jet—SITM—ENDO将合成的siRNA转染入HeLa细胞,以未转染细胞以及错义序列siRNA—scr转染细胞为对照。用细胞计数法检测siRNA对HeLa细胞增殖的影响。流式细胞法检测细胞周期及蛋白表达的变化,RT—PCR法比较转染前后C-myc mRNA表达水平的变化。结果：细胞计数法结果显示,转染24h后c-myc基因siRNA明显抑制MCF-7细胞增殖,转染48h后,抑制效率稳定。c-myc基因siRNA转染后能有效地抑制HeLa细胞的增殖,阻滞细胞周期于G0／G1期,siRNA转染组c-myc mRNA、蛋白的表达量明显低于空白对照组、错义序列组。结论：体外转录合成的siRNA可有效降低HeLa细胞c-myc基因的表达,抑制细胞增殖。     

Role of Toll-like receptors in antisense and siRNA [corrected

About 25 years ago, researchers first demonstrated that a short synthetic oligodeoxynucleotide, referred to as antisense, can inhibit replication of Rous sarcoma virus through hybridization to viral RNA. Since then, several hybridization-based oligonucleotide approaches have been developed to elucidate the functions of genes and their potential as therapeutic agents. Short-interfering (si) RNA is the most recent example. To effectively inhibit gene expression, an antisense or siRNA must be resistant to nucleases, be taken up efficiently by cells, hybridize efficiently with the target mRNA and activate selective degradation of the target mRNA or block its translation without causing undesirable side effects. However, both antisense and siRNA agents have been shown to exert non-target-related biological effects including immune stimulation. Do antisense and siRNA agents work as ligands for Toll-like receptors (TLRs), a family of pathogen-associated, molecular pattern recognition receptors?     

Rigid nanoparticle-based delivery of anti-cancer siRNA: Challenges and opportunities

Gene therapy is a promising strategy to treat various genetic and acquired diseases. Small interfering RNA (siRNA) is a revolutionary tool for gene therapy and the analysis of gene function. However, the development of a safe, efficient, and targetable non-viral siRNA delivery system remains a major challenge in gene therapy. An ideal delivery system should be able to encapsulate and protect the siRNA cargo from serum proteins, exhibit target tissue and cell specificity, penetrate the cell membrane, and release its cargo in the desired intracellular compartment. Nanomedicine has the potential to deal with these challenges faced by siRNA delivery. The unique characteristics of rigid nanoparticles mostly inorganic nanoparticles and allotropes of carbon nanomaterials, including high surface area, facile surface modification, controllable size, and excellent magnetic/optical/electrical properties, make them promising candidates for targeted siRNA delivery. In this review, recent progresses on rigid nanoparticle-based siRNA delivery systems will be summarized.     

靶向HPV16-E6的siRNA对宫颈癌CaSki细胞的抑制作用

以RNA干扰技术为手段,HPV编码的癌蛋白E6为靶标．探讨靶向HPV16-E6的siRNA对宫颈癌细胞生物学行为的影响,并试图阐明该实验的临床意义．构建靶向HPV16-E6的siRNA表达载体,应用体外转染试剂转染HPV16-E6阳性的宫颈癌CaSki细胞．以RT—PCR检测CaSki细胞中E6蛋白的mRNA的表达．借助细胞色素c测定来分析细胞凋亡相关分子的表达和活性．从而研究靶向HPV16-E6的siRNA诱导细胞凋亡的分子机制．RT．PCR检测结果表明,将靶向HPV16-E6的siRNA的表达载体瞬时转染到HPV16-E6阳性的CaSki细胞后,其所舍E6蛋白质和mRNA的表达下调;Westernblotting栓出抑凋亡蛋白Bcl．2的表达亦告下调;细胞色素C释放实验结果显示,HPV16-E6siRNA能够诱导细胞色素c从线粒体释放到细胞浆中。从而诱导细胞凋亡．靶向HPV16-E6的siRNA能够有效抑制细胞增殖并诱导细胞凋亡．靶向HPV16-E6的siRNA为研究重要致瘤蛋白HPV16-E的功能开辟了新途径,给HPV16-E6阳性肿瘤的靶向基因治疗提供新的实验依据．并探索了HPV感染及宫颈癌的新基因疗法．     

siRNA作用效果的靶点依赖性

小分子双链RNA(siRNA)可以高效、特异地沉默目的基因表达 ,为基因功能研究及基因治疗提供了新工具。近年来研究表明针对基因mRNA不同位点随机设计的siRNA在作用效果上存在差异 ,siRNA作用效果有序列依赖性 ,而且与其在基因mRNA上的结合部位的高级结构有关 ,与反义核酸发挥作用的靶点依赖性类似。这一性质对设计高效siRNA为基因功能和基因治疗研究提供指导作用。     

siRNA下调EZH2 基因对肾癌细胞系769-P增殖的影响

目的：运用小干扰RNA下调果蝇zeste 基因增强子人类同源物（enhancer of zeste homolog 2,EZH2）在肾癌细胞系769-P 中 的表达,明确其对肾癌细胞增殖的影响。方法：将处于对数生长期769-P 细胞分为实验组（experiment group）、阴性对照组(negative group)、空白对照组（blank group）,合成靶向EZH2 基因的小干扰RNA片段（EZH2-siRNA）和无效序列片段后,通过脂质体介导分 别转染至实理组和阴性对照组,空白对照组未做任何处理。以qRealtime-PCR 检测EZH2 基因mRNA 水平的变化情况,以MTT 法检测各组细胞增殖变化;流式细胞术（FCM）检测转染后细胞周期变化情况。结果：实理组中EZH2 在mRNA 表达水平明显受 抑制;MTT实验中第4 天始,实验组中769-P 细胞的增殖能力开始受抑制,第5 天时实验组细胞抑制更明显,与阴性对照组和空 白组比较差异有统计学意义（P < 0.05）。siRNA 转染后实验组中G0/G1 期细胞比例明显增多(81.32± 3.14)%,与阴性对照组 (44.13± 1.52)%和空白对照组(45.71± 2.32)%差异有统计学意义。结论：EZH2-siRNA 可有效下调并抑制肾癌细胞769-P的增殖, EZH2在肾癌的发生、发展中发挥了重要作用,为下一步研究肾癌基因治疗提供了理论支持。     

siRNA下调EZH2基因对肾癌细胞系769-P增殖的影响

目的：运用小干扰RNA下调果蝇zeste基因增强子人类同源物（enhancer ofzeste homolog 2,EZH2）在肾癌细胞系769-P中的表达,明确其对肾癌细胞增殖的影响。方法：将处于对数生长期769-P细胞分为实验组（experiment group）、阴性对照组（negative group）、空白对照组（blank group）,合成靶向EZH2基因的小干扰RNA片段（EZH2-siRNA）和无效序列片段后,通过脂质体介导分别转染至实理组和阴性对照组,空白对照组未做任何处理。以qRealtime-PCR检测EZH2基因mRNA水平的变化情况,以MTT法检测各组细胞增殖变化;流式细胞术（FCM）检测转染后细胞周期变化情况。结果：实理组中EZH2在mRNA表达水平明显受抑制;MTT实验中第4天始,实验组中769-P细胞的增殖能力开始受抑制,第5天时实验组细胞抑制更明显,与阴性对照组和空白组比较差异有统计学意义（P〈0.05）。siRNA转染后实验组中G0/G1期细胞比例明显增多（81.32±3.14）%,与阴性对照组（44.13±1.52）%和空白对照组（45.71±2.32）%差异有统计学意义。结论：EZH2-siRNA可有效下调并抑制肾癌细胞769-P的增殖,EZH2在肾癌的发生、发展中发挥了重要作用,为下一步研究肾癌基因治疗提供了理论支持。     

Short interfering RNA (siRNA), a novel therapeutic tool acting on angiogenesis

The formation of new blood vessels, uncontrolled cell expansions and invasions are the common feature of cancer, neovascular inflammatory and ocular diseases, such as age-related macular degeneration (AMD). Short interfering RNA (siRNA) and short-hairpin RNA (shRNA) have recently helped extend our understanding of the mechanisms regulating angiogenesis and tumor developments. Moreover, the early success of these tools has reinforced the therapeutic hopes of preventing endogenous or exogenous gene translation. In vivo experiments using several animal tumor models and human pre-clinical trials augured many benefits to control protein expression and cell signaling. The high specificity of siRNA and shRNA to target a protein is crucial to design a new generation of therapeutic agents. At the present, several investigations are focused on the understanding of both gene function and the proof-of-concept for siRNA-mediated anti-angiogenesis. Taken together, in vitro and in vivo studies shed light on the efficiency of siRNA as a new alternative therapeutic agent.     

Downregulation of MDM2 expression by RNAi inhibits LoVo human colorectal adenocarcinoma cells growth and the treatment of LoVo cells with mdm2siRNA3 enhances the sensitivity to cisplatin

To investigate the biological effect of mdm2 in human colorectal adenocarcinoma LoVo cells, three mdm2siRNA constructions were recombined and transient transfected into human colorectal adenocarcinoma LoVo cells with low differentiation character in vitro. The results showed that mdm2siRNA3 reduced mRNA level of mdm2 and protein level of mdm2, leading to proliferation inhibition on LoVo cells, and reduced tumor growth in nude mice. It was found that depletion of MDM2 in this pattern promoted apoptosis of LoVo cells and Cisplatin (DDP) treated in the mdm2siRNA3 transfected cell population would result in a substantial decrease by MTT colorimetry. Decreasing the MDM2 protein level in LoVo cells by RNAi could significantly inhibit tumor growth both in vitro and in vivo, which indicated that mdm2 gene played a definite role in the development and aggressiveness of human colon carcinoma. It also could be a therapeutic target in colorectal carcinoma. The synergistic activation of RNAi and cell toxicity agents indicated that the combination of chemotherapy and gene therapy will be a promising approach in the future.