1504-siRNA对表达EphA3的肿瘤细胞HGC27的抑制作用

目的:研究原癌基因EphA3的小干扰RNA(siRNA)1504-siRNA对表达EphA3的胃癌细胞HGC27的增殖抑制作用。方法:将携带1504-siRNA的质粒用Vigorous转染试剂瞬时转染HGC27细胞,48 h后收集蛋白,用Western印迹检测EphA3及AKT信号通路中的蛋白分子表达;36 h后收集转染细胞,进行MTT、平板克隆形成和软琼脂克隆形成实验。结果:1504-siRNA能抑制HGC27细胞的EphA3蛋白水平,抑制其增殖、平板克隆形成和软琼脂克隆的形成,抑制pAKT、pmTOR、p-c-Raf、p-4ebp1等信号分子的表达。结论:1504-siRNA可能通过抑制AKT信号通路,而抑制HGC27细胞的增殖、存活能力和恶性程度,它可以作为肿瘤治疗的候选药物。     

Mcl-1在胆盐（GCDA）诱导的肝癌细胞耐药中的作用

目的：探讨抗凋亡蛋白Mcl-1在GCDA诱导的肝癌细胞耐药中的作用及其机制。方法：培养3种肝癌细胞系,用免疫荧光法和Western blot技术检测Mcl-1的表达;GCDA±CYC处理HepG2细胞,采用Western blot技术检测Mcl-1的半衰期变化;用抗癌药物Irinotecan与GCDA对HepG2细胞进行处理,采用MTT法和Western blot技术分别检测细胞增殖抑制率和Mcl-1的表达变化;用RNA干扰技术下调Mcl-1,检测化疗药物对HepG2细胞的敏感性。结果：Mcl-1在肝癌细胞中广泛表达;GCDA能延长Mcl-1的半衰期至6h以上,并明显减弱化疗药物对抗凋亡蛋白Mcl-1的抑制作用,降低癌细胞的药物敏感性;RNA干扰下调Mcl-1能增加癌细胞的药物敏感性。结论：胆盐（GCDA）能诱导HepG2细胞产生耐药性,其作用机制可能是通过延长Mcl-1半衰期增加其蛋白稳定性和抗凋亡作用来促使肝癌细胞抗药的。     

RNA干扰ABCE1基因后可抑制肺癌95-D/NCI-H446细胞的增殖和诱导细胞凋亡

ABCE1作为RNase L抑制剂首先是在脊椎动物中被发现的．前期研究结果显示ABCE1与肺腺癌的发生率及临床分期显著相关．为了进一步研究ABCE1的新功能,构建了ABCE1基因的siRNA表达质粒(RNAi-Ready pSIREN-DNR-DsRed-　Express vector),培养肺癌细胞(95-D和 NCI-H446),用FuGENE 6作为转染试剂转染后,使用荧光显微镜观察转染效果,RT-PCR分析ABCE1基因表达,Western blot 分析ABCE1蛋白的表达,MTT法检测细胞的活性,流式细胞仪分析细胞周期,ELISA法检测细胞凋亡．结果显示：质粒的转染效果较满意,阳性率约为42.70%;在实验组,细胞活性和生长指数明显受到抑制,细胞凋亡明显增加,与对照组比较差异显著(P < 0.05)．上述结果显示,RNA干扰ABCE1基因可显著抑制肺癌细胞(95-D/NCI-H446) RNA的转录、蛋白质的表达,并增加细胞凋亡,为进一步研究ABCE1基因提供必要的基础．     

沉默FNBP1表达诱导7703细胞形态重塑

FNBP1在真核细胞中广泛表达,能通过诱导细胞膜管状化、内陷或变形及后续膜曲率依赖性肌动蛋白聚合过程,参与细胞内吞和运动;还可参与端粒维持及FasL与溶酶体结合过程的调控。研究发现,在肝癌细胞7703中,利用RNA干扰（RNA interference technology,RNAi）技术使内源FNBPI基因表达沉默以后,7703细胞形态发生重塑,由上皮样转变为树状分枝的纤维状,表明FNBP1在细胞形态维持过程中亦具有不可或缺的潜在作用;通过对已知FNBP1蛋白相互作用情况的分析推断,FNBP1可能是通过对肌动蛋白骨架动态组装过程的分子调控参与7703细胞形态的控制。     

shRNA抑制人卵巢癌SW626细胞CXCR4基因的研究

探讨应用RNA干扰技术沉默趋化因子受体4(CXCR4)基因的表达,研究其对人卵巢癌SW626细胞增殖的抑制作用。设计合成两对特异性针对CXCR4基因的Oligo siRNA,用脂质体转染法转染至SW626细胞中,荧光共聚焦显微镜检测转染效率,采用Western blotting检测CXCR4蛋白表达情况,同时利用M'IT试验检测转染后细胞增殖抑制情况。结果显示,转染Oligo siRNA后,SW626细胞CXCR4蛋白表达水平降低(P0.05);细胞增殖的抑制率明显增高(P0.05)。体外合成的特异性针对CXCR4基因的Oligo siRNA对卵巢癌细胞株SW626中CXCR4基因表达和细胞增殖均有明显抑制作用。     

miRNA可抑制肿瘤细胞生长

《科技日报》消息：美国耶鲁大学和奥斯瑞根（Asuragen）公司研究人员发现,一种名为let-7的微小核糖核酸（miRNA）分子,能够持续减缓多种患有不同类型肺癌小鼠模型中的肿瘤生长。这项成果刊登在最近的《细胞周期》（Cellcycle）杂志上。研究人员称,这是关于微小RNA分子可有效治疗包括最致命的肺癌在内的任何癌症的第一篇报道。     

哺乳动物细胞小干扰RNA库的建立和应用

双链小干扰RNA（siRNA）在多种类型细胞中介导特异性的基因沉默,这一现象的发现为深入研究单个基因的功能提供了重要的方法学基础,从而得到了广泛的应用.最近的文献报道了全基因组siRNA库的建立,为高通量基因功能分析和研究提供了新的方法,成为新的研究热点.小干扰RNA库可以用来筛选和研究介导细胞复杂表型和生物学过程的关键基因,通过建立一系列具有目的表型的细胞系,有可能对特定细胞信号调节通路进行更为全面的解析.本文综述了目前在siRNA建库方法方面的进展,并探讨了建立小干扰RNA库中的关键问题.     

下调细胞P21WAF1/CIP1基因表达可抑制单纯疱疹病毒Ⅱ型复制

为了揭示细胞P21蛋白在单纯疱疹病毒Ⅱ型（herpes simplex virus type 2, HSV-2）复制中的作用,通过用HSV-2感染和感染前用特异性小干扰RNA (small interfering RNA,siRNA) 抑制P21基因表达,应用Western 印迹方法检测宿主细胞和病毒蛋白水平,用终点滴定法测定病毒半数组织培养感染量（50% tissue culture infectious dose, TCID50）,以及观察感染细胞的细胞病变效应（cytopathic effect, CPE）等3个方面,揭示细胞P21蛋白水平的变化对病毒复制的影响.结果表明,HSV-2在细胞内复制时可引起P21蛋白水平增高;而用特异性siRNA下调细胞P21基因表达时,可显著地抑制HSV-2 gB蛋白水平,减少培养细胞上清液中病毒TCID50.提示P21蛋白对HSV-2的复制具有重要的作用.     

siRNA对乳腺癌细胞Cyclin E表达和生长抑制作用

研究siRNA对乳腺癌MCF-7细胞株cyclin E表达的抑制及对细胞生长的影响。化学合成针对cyclin E基因的小干扰RNA（siRNA）,转染MCF-7细胞株;分别应用荧光定量PCR和免疫印迹测定cyclin E mRNA和蛋白质的表达,CCK-8测定细胞的增殖活性,流式细胞仪检测细胞周期,软琼脂培养检测细胞克隆形成能力。10、50、100nmol／L siRNA-cyclin E分别使MCF-7细胞cyclin E基因表达降低了24．7％、62．5％和71．0％,蛋白质表达降低了40．8％、66．5％和71．3％。转染siRNA-cyclin E后,G1期细胞增多,S期减少,增殖受到抑制,软琼脂克隆形成率降低。结果提示,在MCF-7细胞株中,导入针对cyclin E的siRNA,可有效抑制cyclin E的表达,进而使细胞增殖减缓,逆转其恶性表型。     

STAT3-siRNA对人结直肠癌细胞的干涉作用

目的:研究STAT3-siRNA对STAT3基因表达阳性的结直肠癌细胞凋亡的影响。方法:应用脂质体转染试剂将STAT3-siRNA表达盒(STAT3-siRNA expression cassettes,STAT3-SECs)体外转染至人结直肠癌SW480细胞及人成纤维细胞中,同时分别设立人成纤维对照组、SW480对照组、SW480错配链-SECs组和SW480空转染试剂组。于48h后收集细胞,先经荧光染色方法观察细胞表象变化,再通过流式细胞仪检测人结直肠癌SW480细胞凋亡情况,后分别提取细胞总RNA,用RT-PCR测定STAT3基因在mRNA水平的表达。结果:SW480STAT3-SECs组的细胞可见凋亡小体,出现明显的凋亡现象,而人成纤维对照组、人成纤维STAT3-SECs组、SW480对照组、SW480错配链-SECs组和SW480空转染试剂组未出现明显的凋亡现象。SW480STAT3-SECs组细胞的凋亡比率较SW480对照组、SW480错配链-SECs组和SW480空转染试剂组有明显的增高。RT-PCR所得数据经统计学处理得出:SW480STAT3-SECs组细胞的STAT3基因表达在mRNA水平上显著低于SW480对照组(P0.01);而人成纤维对照组与人成纤维STAT3-SECs组,SW480细胞对照组与SW480错配链-SECs组、SW480空转染试剂组之间无明显差异(P0.05)。结论:应用RNAi技术沉默STAT3基因可以降低人结直肠癌SW480细胞中STAT3的表达,诱导细胞的凋亡。     

A comparison of siRNA efficacy predictors

Short interfering RNA (siRNA) efficacy prediction algorithms aim to increase the probability of selecting target sites that are applicable for gene silencing by RNA interference. Many algorithms have been published recently, and they base their predictions on such different features as duplex stability, sequence characteristics, mRNA secondary structure, and target site uniqueness. We compare the performance of the algorithms on a collection of publicly available siRNAs. First, we show that our regularized genetic programming algorithm GPboost appears to have a higher and more stable performance than other algorithms on the collected datasets. Second, several algorithms gave close to random classification on unseen data, and only GPboost and three other algorithms have a reasonably high and stable performance on all parts of the dataset. Third, the results indicate that the siRNAs' sequence is sufficient input to siRNA efficacy algorithms, and that other features that have been suggested to be important may be indirectly captured by the sequence.     

Clinical advances of siRNA therapeutics

Small interfering RNA (siRNA) enables efficient target gene silencing by employing a RNA interference (RNAi) mechanism, which can compromise gene expression and regulate gene activity by cleaving mRNA or repressing its translation. Twenty years after the discovery of RNAi in 1998, ONPATTRO? (patisiran) (Alnylam Pharmaceuticals, Inc.), a lipid formulated siRNA modality, was approved for the first time by United States Food and Drug Administration and the European Commission in 2018. With this milestone achievement, siRNA therapeutics will soar in the coming years. Here, we review the discovery and the mechanisms of RNAi, briefly describe the delivery technologies of siRNA, and summarize recent clinical advances of siRNA therapeutics.     

The potential therapeutic of siRNA eye drops in ocular diseases

In recent years, researchers have expressed an ongoing interest in developing RNA interference (RNAi) technology for therapeutic gene suppression in various diseases. Preclinical studies in animal models and cultured cell studies indicated that RNAi technology was an effective experimental tool against a variety of ocular diseases, and some small interference RNA (siRNA) drugs have been entered into clinical trials in Stage I and Stage II. However, in these studies siRNAs were delivered into ocular tissues via either systemic or subconjunctival/intravitreous injection, which is invasive and harmful if repeated. Based on this evidence, we hypothesize that topical application of siRNA eye drops may be a safe and effective therapeutic option in ocular surface diseases with temporary changes of gene expression. Furthermore, siRNA eye drops targeting different genes may simultaneously treat several ocular surface diseases.     

Local RNA target structure influences siRNA efficacy: systematic analysis of intentionally designed binding regions

Contradictory reports in the literature have emphasised either the sequence of small interfering RNAs (siRNA) or the structure of their target molecules to be the major determinant of the efficiency of RNA interference (RNAi) approaches. In the present study, we analyse systematically the contributions of these parameters to siRNA activity by using deliberately designed mRNA constructs. The siRNA target sites were included in well-defined structural elements rendering them either highly accessible or completely involved in stable base-pairing. Furthermore, complementary sequence elements and various hairpins with different stem lengths and designs were used as target sites. Only one of the strands of the siRNA duplex was found to be capable of silencing via its respective target site, indicating that thermodynamic characteristics intrinsic to the siRNA strands are a basic determinant of siRNA activity. A significant obstruction of gene silencing by the same siRNA, however, was observed to be caused by structural features of the substrate RNA. Bioinformatic analysis of the mRNA structures suggests a direct correlation between the extent of gene-knockdown and the local free energy in the target region. Our findings indicate that, although a favourable siRNA sequence is a necessary prerequisite for efficient RNAi, complex target structures may limit the applicability even of carefully chosen siRNAs.     

siRNA纳米递送系统研究进展

小干扰RNA (small interfering RNA,siRNA)是RNA干扰的引发物,激发与之互补的目标mRNA沉默,对基因调控及疾病治疗有重要意义。siRNA作为药物需要克服血管屏障、实现细胞内吞及溶酶体逃逸,同时还需要避免核酸酶作用下发生降解。因此,设计合适的纳米载体以帮助siRNA成功递送进细胞并发挥作用是目前siRNA药物发展的重要目标。纳米载体的材料种类、尺寸、结构、表面修饰等精确设计是实现siRNA药物成功递送的重要因素。随着研究的深入和应用的发展,siRNA药物纳米载体的精确控制制备、精准靶向递送及多功能化取得了较好的成果。本文围绕siRNA药物纳米载体,对siRNA药物应用及其递送困难、siRNA药物纳米载体主要设计策略、目前siRNA药物上市情况进行介绍,同时对其未来发展方向进行展望。     

Selection and optimization of asymmetric siRNA targeting the human c-MET gene

The silencing of specific oncogenes via RNA interference (RNAi) holds great promise for the future of cancer therapy. RNAi is commonly carried out using small interfering RNA (siRNA) composed of a 19 bp duplex region with a 2-nucleotide overhang at each 3′ end. This classical siRNA structure, however, can trigger non-specific effects, which has hampered the development of specific and safe RNAi therapeutics. Previously, we developed a novel siRNA structure, called asymmetric shorter-duplex siRNA (asiRNA), which did not cause the non-specific effects triggered by conventional siRNA, such as off-target gene silencing mediated by the sense strand. In this study, we first screened potent asiRNA molecules targeting the human c-MET gene, a promising anticancer target. Next, the activity of a selected asiRNA was further optimized by introducing a locked nucleic acid (LNA) to maximize the gene silencing potency. The optimized asiRNA targeted to c-MET may have potential as a specific and safe anticancer RNAi therapeutic.     

.限制性siRNA干涉文库的构建以及影响HeLa细胞增殖新功能基因的扫描

利用RNA干涉文库进行大规模高通量的功能基因扫描,已成为发现新功能基因的重要方式和手段．为了寻找在细胞增殖和分化过程中的新功能基因,根据斯坦福大学公布的与人类胚胎干细胞和造血干细胞增殖和分化过程中有关基因的基因芯片的分析结果,组建了与细胞增殖和分化有关的RNA限制性干涉文库．该文库包括靶向各类基因的载体,如包括转录因子、各类蛋白激酶、细胞周期调控蛋白以及一些未知功能基因在内的225个基因．利用这个限制性RNA干涉文库对控制HeLa细胞增殖的基因进行筛选．并通过WST-1高通量检测,发现了2个同HeLa细胞增殖相关的基因：CNKSR3(Homo sapiens CNKSR family member 3)和Fosl2 Homo sapiens FOS like antigen 2),并初步证实：沉默CNKSR3会促进HeLa细胞的增殖,而沉默Fosl2则抑制HeLa细胞的增殖功能．     

Initial evidence of functional siRNA machinery in dinoflagellates

Dinoflagellates are a major group of protists widely distributed in the aquatic environments. Many species in this lineage are able to form harmful algal blooms (HAB), some even producing toxins, making this phylum the most important contributors of HAB in the marine ecosystem. Despite the ecological importance, the molecular mechanisms underpinning the basic biology and HAB formation of dinoflagellates are poorly understood. While the high-throughput sequencing studies have documented a large and growing number of genes in dinoflagellates, their functions remained to be experimentally proven using a functional genetic tool. Unfortunately, no such tool is yet available. This study was aimed to adopt the RNA interference (RNAi) gene-silencing tool for dinoflagellate research, and to investigate the potential effects of RNAi-based silencing of proton-pump rhodopsin and CO₂-fixing enzyme Rubisco encoding genes in dinoflagellates. It was found that RNAi treatment caused a significant decrease in growth rate in both species. Compared with the non- endogenous target (GFP-siRNA) and the blank control, RNAi treatments also suppressed the expression of the target genes. These results constitute the first experimental evidence of the existence and operation of siRNA in two species of dinoflagellates, present initial evidence that dinoflagellate rhodopsins are functional as a supplemental energy acquisition mechanism, and provide technical information for future functional genetic research on dinoflagellates.     

Knockdown of Ki-67 by small interfering RNA leads to inhibition of proliferation and induction of apoptosis in human renal carcinoma cells

To investigate the effect of small-interfering RNA (siRNA) targeted against Ki-67, which is an attractive molecular target for cancer therapy, on inhibiting Ki-67 expression and cell proliferation in human renal carcinoma cells (HRCCs), siRNAs were used to inhibit the expression of Ki-67 in HRCCs. Ki-67 mRNA levels were detected by RT-PCR and in situ hybridization analysis. Ki-67 protein levels were detected by Western blot and immunocytochemistry analysis. TUNEL assay was used to measure the apoptosis of carcinoma cells. Results of RT-PCR and in situ hybridization demonstrated reduction of Ki-67 mRNA expression in Ki-67 siRNAs treated 786-0 cells. Similar reduction in Ki-67 protein measured by Western blot and immunocytochemistry was observed in cells transfected with Ki-67 siRNA. Ki-67-siRNA treatment of HRCCs resulted in specific inhibition of proliferation and increased apoptotic cell death. From these findings we conclude that inhibition of Ki-67 expression by siRNA may be a reasonable approach in renal cancer therapy.     

Competition potency of siRNA is specified by the 5'-half sequence of the guide strand

Small-interfering RNAs (siRNAs) execute specific cellular gene silencing by exploiting the endogenous RNA interference (RNAi) pathway. Therefore, excess amounts of siRNAs can saturate cellular RNAi machineries. Indeed, some siRNAs saturate the RNA-induced silencing complex (RISC) and competitively inhibit silencing by other siRNAs. However, the molecular feature of siRNAs that specifies competition potency has been undetermined. While previous reports suggested a correlation between the competition potency and silencing efficiency of siRNAs, we found that the silencing efficiency was insufficient to explain the competition potency. Instead, we show that the nucleotide sequence of the 5′-half of the guide strand determines the competition potency of an siRNA. Our finding provides important information for understanding the mechanistic basis of competition in combinatorial RNAi treatment.