发夹RNA(shRNA)在哺乳动物RNAi研究中的应用

在哺乳动物的RNAi研究中,载体表达的shRNA分子比细胞同时表达的siRNA分子的正义链与反义链对靶基因的抑制效率要高。shRNA可由PolⅢ的启动子在体内表达产生,酶切cDNA和shRNA芯片是产生shRNA的最新方法。对shRNA的设计应注意靶基因序列、环序列以及载体酶切位点的选择。诱导表达shRNA的载体系统的表达效率有所差异,质粒载体转染效率尚不稳定,且持续时间短,通过病毒载体介导是目前进行基因敲除最有效的工具。     

Optimization of short hairpin RNA for lentiviral-mediated RNAi against WAS

The expression of short hairpin RNAs (shRNAs) with lentiviral vectors is useful to induce stable RNA interference, particularly in hematopoietic cells. Since primary cells integrate few copies of vector, we tested if several shRNA cassette modifications could improve knock-down efficacy. Using two shRNA sequences previously shown to inhibit the human WAS gene expression, we found that neither increasing the shRNA stem length from 19-nt to 29-nt, nor modifying the loop with 4-nt, 9-nt artificial loops or with the mir30 loop improved vector-induced shRNA efficacies. This cautions against extrapolating results obtained with synthetic molecules to shRNAs that are stably expressed from viral vectors. On the other hand, the duplication of the shRNA expression cassette resulted in twice as much knock-down per copy of integrated vector. This strategy allowed a strong suppression of WASp in CD34⁺ cells and will facilitate future studies on the role of WASp in human cells.     

Fluorescence-based evaluation of shRNA efficacy

RNA interference is a cellular mechanism regulating levels of mRNAs. It has been widely exploited to knock down specific protein targets. The selected interfering RNA sequence greatly influences its ability to knock down the target. Here we present a method for constructing multiple testing plasmids which express small hairpin RNAs (shRNA) targeting different regions of an mRNA. A simple fluorescence test in cultured cells allows convenient evaluation of mRNA knockdown by many different shRNAs on 96-well plates. We show that software predicted shRNAs have varying efficacies and only 2 of the 7 tested shRNAs significantly knocked down their targets.     

Drosophila U6 promoter-driven short hairpin RNAs effectively induce RNA interference in Schneider 2 cells

The effect of RNA interference (RNAi) is generally more potent in Drosophila Schneider 2 (S2) cells than in mammalian cells. In mammalian cells, PolIII promoter-based DNA vectors can be used to express small interfering RNA (siRNA) or short hairpin RNA (shRNA); however, this has not been demonstrated in cultured Drosophila cells. Here we show that shRNAs transcribed from the Drosophila U6 promoter can efficiently trigger gene silencing in S2 cells. By targeting firefly luciferase mRNA, we assessed the efficacy of the shRNAs and examined the structural requirements for highly effective shRNAs. The silencing effect was dependent on the length of the stem region and the sequence of the loop region. Furthermore, we demonstrate that the expression of the endogenous cyclin E protein can be repressed by the U6 promoter-driven shRNAs. Drosophila U6 promoter-based shRNA expression systems may permit stable gene silencing in S2 cells.     

Lentiviral vectors that carry anti-HIV shRNAs: problems and solutions

BACKGROUND: HIV-1 replication can be inhibited with RNA interference (RNAi) by expression of short hairpin RNA (shRNA) from a lentiviral vector. Because lentiviral vectors are based on HIV-1, viral sequences in the vector system are potential targets for the antiviral shRNAs. Here, we investigated all possible routes by which shRNAs can target the lentiviral vector system. METHODS: Expression cassettes for validated shRNAs with targets within HIV-1 Leader, Gag-Pol, Tat/Rev and Nef sequences were inserted in the lentiviral vector genome. Third-generation self-inactivating HIV-1-based lentiviral vectors were produced and lentiviral vector capsid production and transduction titer determined. RESULTS: RNAi against HIV-1 sequences within the vector backbone results in a reduced transduction titer while capsid production was unaffected. The notable exception is self-targeting of the shRNA encoding sequence, which does not affect transduction titer. This is due to folding of the stable shRNA hairpin structure, which masks the target for the RNAi machinery. Targeting of Gag-Pol mRNA reduces both capsid production and transduction titer, which was improved with a human codon-optimized Gag-Pol construct. When Rev mRNA was targeted, no reduction in capsid production and transduction titer was observed. CONCLUSIONS: Lentiviral vector titers can be negatively affected when shRNAs against the vector backbone and the Gag-Pol mRNA are expressed during lentiviral vector production. Titer reductions due to targeting of the Gag-Pol mRNA can be avoided with a human codon-optimized Gag-Pol packaging plasmid. The remaining targets in the vector backbone may be modified by point mutations to resist RNAi-mediated degradation during vector production.     

Interference RNA (RNAi)-based silencing of endogenous thrombopoietin receptor (Mpl) in Dami cells resulted in decreased hNUDC-mediated megakaryocyte proliferation and differentiation

Recently our laboratory reported evidence showing that hNUDC acts as an additional cytokine for thrombopoietin receptor (Mpl). Previously known as the human homolog of a fungal nuclear migration protein, hNUDC plays a critical role in megakaryocyte differentiation and maturation. Here we sought to further clarify the hNUDC-Mpl ligand-receptor relationship by utilizing interference RNA (RNAi) to knockdown Mpl expression in a megakaryocyte cell line. We created U6 promoter driven constructs to express short hairpin RNAs (shRNA) with affinity for different sites on Mpl mRNA. By including Mpl-EGFP fusion protein in these constructs, we were able to effectively screen the shRNA that was most efficient in inhibiting Mpl mRNA expression. This shRNA was subsequently transferred into a lentivirus vector and transduced into Dami cells, a cell line which constitutively expresses endogenous Mpl. This lentiviral vector was also designed to simultaneously express EGFP to monitor transfection efficiency. Our results show that lentivirus can be used to effectively deliver shRNAs into Dami cells and cause specific inhibition of Mpl protein expression after transduction. Furthermore, we show the functional effects of shRNA-mediated Mpl silencing by demonstrating reduced hNUDC stimulated megakaryocyte proliferation and differentiation. Thus, the use of a RNAi knockdown strategy has allowed us to pinpoint the connection of hNUDC with Mpl in the regulation of megakaryocyte maturation.     

Multiple shRNA-mediated knockdown of TACE reduces the malignancy of HeLa cells

Tumor necrosis factor-alpha (TNF-α) converting enzyme (TACE) is a key enzyme involved in the proteolytic shedding of the ectodomain of several membrane-bound growth factors, cytokines and receptors. Here, we constructed a multiple short hairpin RNA (shRNA) expression vector containing four shRNAs against TACE. We found that in HeLa cells our multiple shRNAs vector produced a higher level of TACE knockdown than any single shRNA vector containing only one TACE shRNA. Silencing TACE expression in HeLa cells decreased their malignancy by decreasing the proliferation, adhesion and migration, as well as inducing apoptosis in these cells. Furthermore, our data suggest that the effects of TACE on the malignancy of HeLa cells may be mediated via activation of the EGFR (epidermal growth factor receptor) signaling pathway. Our findings suggest that using a combination of shRNAs within one vector to silence the expression of TACE might be a potential therapeutic strategy for tumors.     

Dicer-independent processing of short hairpin RNAs

Short hairpin RNAs (shRNAs) are widely used to induce RNA interference (RNAi). We tested a variety of shRNAs that differed in stem length and terminal loop size and revealed strikingly different RNAi activities and shRNA-processing patterns. Interestingly, we identified a specific shRNA design that uses an alternative Dicer-independent processing pathway. Detailed analyses indicated that a short shRNA stem length is critical for avoiding Dicer processing and activation of the alternative processing route, in which the shRNA is incorporated into RISC and processed by the AGO2-mediated slicer activity. Such alternatively processed shRNAs (AgoshRNAs) yield only a single RNA strand that effectively induces RNAi, whereas conventional shRNA processing results in an siRNA duplex of which both strands can trigger RNAi. Both the processing and subsequent RNAi activity of these AgoshRNAs are thus mediated by the RISC-component AGO2. These results have important implications for the future design of more specific RNAi therapeutics.     

The reversion effect of the RNAi-silencing mdr1 gene on multidrug resistance of the leukemia cell HT9

Overexpression of P-glycoprotein (P-gp), the mdr1 gene product, confers multidrug resistance (MDR) to tumor cells and often limits the efficacy of chemotherapy. This study evaluated RNAi for specific silencing of the mdr1 gene and reversion of multidrug resistance. Three different short hairpin RNAs (shRNAs) were designed and constructed in a pSilencer 3.1-H1 neo plasmid. The shRNA recombinant plasmids were transfected into HT9 leukemia cells. The RNAi effect was evaluated by real-time PCR, Western blotting and cell cytotoxicity assay. In the cell, shRNAs can specifically down-regulate the expression of mdr1, mRNA and P-gp. Resistance against harringtonine, doxorubicin and curcumin was decreased. The study indicated that shRNA recombinant plasmids could modulate MDR in vitro.     

用于筛选shRNA的新型双萤光素酶报告基因载体的构建及应用

目的:构建一个新型双萤光素酶报告基因载体用于准确高效地筛选有效抑制靶基因表达的shRNA。方法:采用PCR、定向克隆、基因重组等分子生物学方法,将双萤光素酶报告基因系统中需要的报告基因载体、shRNA真核表达载体和内参载体三个功能,整合于一个新型的双萤光素酶报告基因载体pFLuc-C-TK-RLuc-shRNA之中。应用该载体对靶向人PD1基因以及Furin基因的shRNA进行干涉效果比较。结果:应用p FLuc-C-TK-RLuc-shRNA载体进行单质粒转染方法与传统的三质粒转染方法均提示shRNA#1对靶基因PD1的抑制效果最优;但是使用新型双萤光素酶报告基因载体检测结果的标准差数值显著低于传统方法。以pFLuc-C-TK-RLuc-shRNA单质粒转染方法得到的各组样品结果的均一性显著提高,能够准确地反映出shRNA#3较shRNA#2具有更强的Furin基因抑制作用;而传统方法未能有效判断二者的差异。结论:新型双萤光素酶报告基因载体简化了操作步骤,降低了传统三质粒共转染方法所引起的检测结果大幅波动,进一步增强了双萤光素酶报告基因系统的信噪比,提高了筛选抑制靶基因表达shRNA的准确性。     

Short hairpin RNAs against eotaxin or interleukin‐5 decrease airway eosinophilia and hyper‐responsiveness in a murine model of asthma

Background

Eosinophilia plays the major role in the pathogenesis of asthma and correlates with the up‐regulation of eotaxin, which, together with interleukin (IL)‐5, is important for differentiation, chemo‐attraction, degranulation, and survival of eosinophils in local tissue. In a previous study, we found that administration of lentivirus‐delivered short hairpin RNA (shRNA) to suppress the expression of IL‐5 inhibited airway inflammation. The present study aimed to investigate the role of eotaxin shRNA and the synergistic effect of eotaxin and IL‐5 shRNAs on airway inflammation in an ovalbumin (OVA)‐induced murine model of asthma.

Methods

Lentivirus‐delivered shRNAs were used to suppress the expression of eotaxin and/or IL‐5 in local tissue in an OVA‐induced murine asthma model.

Results

Intra‐tracheal administration of lentivirus containing eotaxin shRNA expressing cassette (eoSEC3.3) efficiently moderated the characteristics of asthma, including airway hyper‐responsiveness, cellular infiltration of lung tissues, and eotaxin and IL‐5 levels in bronchio‐alveolar lavage fluid. Administration of lentiviruses expressing IL‐5 or eotaxin shRNAs (IL5SEC4 + eoSEC3.3) also moderated the symptoms of asthma in a mouse model.

Conclusions

Local delivery of lentiviruses expressing IL‐5 and eotaxin shRNAs provides a potential tool in moderating airway inflammation and also has the potential for developing clinical therapy based on the application of shRNAs of chemokines and cytokines involved in T helper 2 cell inflammation and eosinophilia. Copyright © 2008 John Wiley & Sons, Ltd.     

Conditional knockdown of hMRS2 results in loss of mitochondrial Mg(2+) uptake and cell death

The human gene MRS2L encodes a mitochondrial protein distantly related to CorA Mg²⁺ transport proteins. Constitutive shRNA-mediated knockdown of hMRS2 in human HEK-293 cell line was found here to cause death. To further study its role in Mg²⁺ transport, we have established stable cell lines with conditionally expressing shRNAs directed against hMRS2L . The cells expressing shRNA for several generations exhibited lower steady-state levels of free mitochondrial Mg²⁺ ([Mg²⁺]_m) and reduced capacity of mitochondrial Mg²⁺ uptake than control cells. Long-term expression of shRNAs resulted in loss of mitochondrial respiratory complex I, decreased mitochondrial membrane potential and cell death. We conclude that hMrs2 is the major transport protein for Mg ⁺ uptake into mitochondria and that expression of hMrs2 is essential for the maintenance of respiratory complex I and cell viability.     

抑制人cFLIP 基因表达的shRNA 质粒的构建与功能验证

目的:构建特异性抑制cFLIP基因表达的质粒并检测其对肝癌细胞的影响。方法:根据人cFLIP mRNA的序列,设计合成3对cFLIP基因的shRNA,将其连入干扰载体,转染HepG2,蛋白印迹法检测基因表达情况,以检测其对肝癌细胞的影响。结果:构建了特异性抑制肝癌细胞中cFLIP表达的质粒。结论:成功构建能特异且高效阻断cFLIP表达的shRNA表达质粒,为进一步研究cFLIP基因对肝癌增殖的影响及其临床应用奠定了基础。     

Functional Selection of shRNA Loops from Randomized Retroviral Libraries

Gene silencing by RNA interference (RNAi) can be achieved by the ectopic expression of tailored short hairpin RNAs (shRNAs) which after export to the cytoplasm are processed by Dicer and incorporated into the RNA induced silencing complex (RISC). Design rules for shRNAs have been the focus of several studies, but only a few reports have turned the attention to the sequence of the loop-region. In this work we selected high-functional and low-functional shRNA loops from retroviral hairpin-loop-libraries in an RNAi reporter assay. The procedure revealed a very significant and stem sequence-dependent effect of the loop on shRNA function and although neither strong consensus loop sequence nor structural motifs could be identified, a preferred loop sequence (5'-UGUGCUU-3') was found to support robust knock down with little stem sequence dependency. These findings will serve as a guide for designing shRNAs with improved knock down capacity.     

RNA干扰抑制麻疹病毒体外复制的实验研究

为了研究短发夹RNA(shRNA)介导的RNA干扰对麻疹病毒体外复制的抑制作用,构建靶向与麻疹病毒复制密切相关的宿主细胞基因Rab9 GTPase基因特异性shRNA表达载体,分别转染Vero-E6和B95a细胞后感染麻疹病毒Edmonston株和野生株。逆转录聚合酶链反应(RT-PCR)和免疫印迹技术(Western-blot)检测转染细胞内Rab9 GTPase基因表达水平;标准蚀斑试验测定麻疹病毒滴度。结果显示转染细胞内Rab9 GTPase mRNA和蛋白质的表达水平同对照组相比明显降低,标准蚀斑试验显示麻疹病毒的复制受到显著抑制,抑制率达到90%以上。结果表明载体介导的shRNAs能通过特异性下调Rab9 GTPase基因表达抑制麻疹病毒体外复制,Rab9 GTPase可能成为治疗麻疹病毒感染的RNA干扰靶。     

Evaluation of Interferon Response Induced by Anti-Myostatin shRNA Constructs in Goat (Capra hircus) Fetal Fibroblasts by Quantitative Real TIME-Polymerase Chain Reaction

RNAi is an evolutionary conserved, highly efficient, and cost effective technique of gene silencing. It holds considerable promise and success has been achieved both in vitro and in vivo experiments. However, it is not devoid of undesirable side effects as dsRNA can trigger the immune response and can also cause non-specific off-target gene silencing. In the present study, silencing of myostatin gene, a negative regulator of myogenesis, was evaluated in caprine fetal fibroblasts using three different shRNA constructs. Out of these three constructs, two constructs sh1 and sh2 showed, 72% and 50% reduction (p < 0.05) of myostatin mRNA, respectively. Efficient suppression (42–86%) of MSTN gene (p < 0.05) was achieved even by reducing the concentration of shRNA constructs. The induction of classical interferon stimulated gene (Oligoandenylate Synthetase-1, OAS-1) was studied to analyze the immune response against shRNAs. Notably, a reduction in the potency of shRNAs to induce interferon response was observed at lower concentration for OAS1 gene. The results obtained in the study would be helpful in the abrogation of the bystander effects of RNAi for long term stable expression of anti-MSTN expression constructs in the muscle.     

In vitro inhibition of transmissible gastroenteritis coronavirus replication in swine testicular cells by short hairpin RNAs targeting the ORF 7 gene

ABSTRACT: BACKGROUND: Transmissible gastroenteritis (TGE) is a highly contagious viral disease of swine, characterized by severe vomiting, diarrhea, and high mortality. Currently, the vaccines for it are only partially effective and no specific drug is available for treatment of TGE virus (TGEV) infection. RNA interference has been confirmed as a new approach for controlling viral infections. In this study, the inhibitory effect of short hairpin RNAs (shRNAs) targeting the ORF 7 gene of TGEV on virus replication was examined. RESULTS: Four theoretically effective sequences of TGEV ORF 7 gene were designed and selected for construction of shRNA expression plasmids. In the reporter assays, three of four shRNA expression plasmids were able to inhibit significantly the expression of ORF 7 gene and replication of TGEV, as shown by real-time quantitative RT-PCR analysis of viral ORF 7 and N genes and detection of virus titers (TCID50/ml). Stable swine testicular (ST) cells expressing the shRNAs were established. Observation of the cytopathic effect and apoptosis, as well as a cell proliferation assay demonstrated that the three shRNAs were capable of protecting ST cells against TGEV destruction, with high specificity and efficiency. CONCLUSIONS: Our results indicated that plasmid-transcribed shRNAs targeting the ORF 7 gene in the TGEV genome effectively inhibited expression of the viral target gene and viral replication in vitro. These findings provide evidence that the shRNAs have potential therapeutic application for treatment of TGE.