Poly(beta-amino ester) as a carrier for si/shRNA delivery in lung cancer cells

Efficient delivery of small interfering RNA (siRNA) or small hairpin RNA (shRNA) is a critical concern in RNA interference (RNAi) studies. In the present study, we evaluated biodegradable poly(beta-amino ester) (PAE) carrier composed of low molecular weight polyethylenimine and poly(ethylene glycol) for si/shRNA delivery in lung cancer cells. PAE carrier successfully delivered EGFP (enhanced green fluorescence protein) siRNA (siGFP) and silenced EGFP expression. The silencing achieved with PAE carrier was found to be nearly 1.5 times superior and safer than standard PEI25K. Also, our PAE carrier exhibited superior Akt1 shRNA delivery (shAkt) and thereby silenced oncoprotein Akt1 efficiently. PAE-shAkt mediated Akt1 knock-down hindered cancer cell growth in Akt1 specific manner. Superior shAkt delivery and low cytotoxicity of PAE carrier promoted Akt1 knock-down specific apoptosis, while low delivery efficiency and high cytotoxicity of PEI25K carrier mainly exhibited undesirable necrosis. Moreover, basic cancer properties like cell proliferation, malignancy and metastasis were reduced more efficiently using PAE-shAkt system. These findings demonstrated the potential of PAE as an alternative to PEI25K in si/shRNA-based RNAi studies.     

Small interfering RNAs inhibit infectious bursal disease virus replication in Vero cells

Small interfering RNA (siRNA) molecules are considered to be a promising antiviral therapeutics. This study was performed to analyze the application of siRNA against infectious bursal disease virus (IBDV) replication. Two siRNAs were designed to target common coding sequences of four IBDV proteins. Corresponding vectors were constructed to express anti-IBDV short hairpin RNAs (shRNA) that were tested for their antiviral effect in Vero cells. The results showed that expressed shRNA inhibited the virus replication to a significant extent (92%) as determined by the virus titration in cell culture. This outcome demonstrated the effectiveness of RNA interference (RNAi) based mechanism against the IBDV in vitro.     

The suppression of lung tumorigenesis by aerosol-delivered folate–chitosan-graft-polyethylenimine/Akt1 shRNA complexes through the Akt signaling pathway

RNA interference (RNAi) represents a promising new approach to the inhibition of gene expression in vitro and in vivo, and has therapeutic potential for human diseases. Efficient delivery of small interfering RNA (siRNA) or small hairpin RNA (shRNA) is a critical concern in RNAi studies. Here we report the development of a new polymeric gene carrier for cancer cell-targeting, designed to enhance the intracellular delivery of shRNA and reduce cytotoxicity. Folate–chitosan-graft-polyethylenimine (FC-g-PEI) copolymer was prepared by an imine reaction between periodate-oxidized folate–chitosan (FC) and low molecular weight polyethylenimine (PEI). FC-g-PEI copolymer was investigated as a potential cancer cell-targeting gene carrier. The composition of FC-g-PEI was characterized using ¹H nuclear magnetic resonance (¹H NMR), and particle size and zeta potential of FC-g-PEI/shRNA complexes were measured using dynamic light scattering (DLS). FC-g-PEI showed good shRNA condensation ability and high protection of shRNA from nuclease attack. It also exhibited lower cytotoxicity compared to PEI 25K control, and showed good cancer cell-targeting ability. Furthermore, aerosol delivery of FC-g-PEI/Akt1 shRNA complexes suppressed lung tumorigenesis in a urethane-induced lung cancer model mouse through the Akt signaling pathway. Together, these results suggest that FC-g-PEI may be useful for shRNA-based gene therapy.     

脯氨酰羟化酶RNA干扰慢病毒载体的构建与鉴定

背景：慢病毒载体可稳定介导基因沉默且具有较高的转染效率。 目的：构建并鉴定脯氨酰羟化酶 RNA干扰慢病毒载体。 方法：针对脯氨酰羟化酶2基因序列设计RNA干扰靶序列,合成靶序列的寡聚DNA,退火形成双链DNA,与经Age Ⅰ和EcoR Ⅰ酶切的pGCSIL-GFP连接、转化大肠杆菌感受态细胞,产生重组RNA干扰慢病毒表达载体,PCR筛选阳性克隆,测序鉴定。 结果与结论：PCR和DNA测序证实合成的含脯氨酰羟化酶短发卡RNA慢病毒载体寡核苷酸链正确插入pGCSIL-GFP载体。说明实验成功构建脯氨酰羟化酶基因RNA干扰慢病毒载体。     

The development of vector constructions for respiratory syncitial virus (RSV) P-gene silencing

Interfering RNA (RNAi) is a powerful tool to silence gene expression on the level of mRNA. To knock-down gene expression by using RNAi two major methods of mRNA silencing exist. First method utilizes siRNA (small interfering RNA), a readily processed dsRNA, that enters RISC complex and destroy target mRNA after transfection into the cells. The second method based on the construction of plasmid DNA that expresses shRNA (short harpin RNA) from U6 or CMV promoter. shRNA gets processed by Drosha and Dicer RNAses inside the cell before it translocates to the cell cytoplasm and affects the level of target RNA. In this study we modified lentiviral vector pGIPZ expressing tFP-IRES-Puro-shRNA(mir30) cassette by introducing BamH I restriction site downstream of this cassette. This modification makes possible to clone specific shRNA sequences in pGIPZ vector using XhoI/BamHI restriction sites instead of the original recombination. Three shRNAs against phosphoprotein P of respiratory sinthitial virus (RSV) and shRNA against human CD43 as a control were generated and cloned into modified so-called pCIPD vector. Monkey kidney cells MA-104 were stably transduced with four shRNA constructs. In conclusion, the generated lentiviral vector pCIPD can be successfully used for efficient gene silencing and virus replication in a broad variety of cells.     

PTTG基因siRNA载体的构建与鉴定

目的构建针对垂体腺瘤细胞系AtT20的高效率沉默PTTG基因的shRNA表达载体。方法合成特异性干扰PTTG基因的小发卡RNA(shRNA)片段,并与pGenesil2载体连接,构建PTTG干扰载体(pGenesil2-PTTG shRNA)。利用脂质体将其转染AtT20细胞,分为正常细胞对照组、阴性对照组和3个shRNA干扰组(pGenesil2-PTTG siRNA1、2及3),应用半定量逆转录聚合酶链式反应(RT-PCR)和Western blot法分析转染后AtT20细胞中PTTG的mRNA和蛋白表达水平。结果酶切证实PTTG-shRNA表达载体构建成功;转染效率可达75%左右。转染pGenesil2-PTTG shRNA后,3个干扰组的AtT20细胞中PTTG的mRNA和蛋白表达水平均较正常对照组显著降低(P<0.01)。结论成功构建了能高效沉默PTTG的RNAi表达载体;且pGenesil2-PTTG siRNA高效地抑制了垂体腺瘤细胞AtT20细胞中PTTG綦因的表达。     

Shortcomings of short hairpin RNA-based transgenic RNA interference in mouse oocytes

Background

RNA interference (RNAi) is a powerful approach to study a gene function. Transgenic RNAi is an adaptation of this approach where suppression of a specific gene is achieved by expression of an RNA hairpin from a transgene. In somatic cells, where a long double-stranded RNA (dsRNA) longer than 30 base-pairs can induce a sequence-independent interferon response, short hairpin RNA (shRNA) expression is used to induce RNAi. In contrast, transgenic RNAi in the oocyte routinely employs a long RNA hairpin. Transgenic RNAi based on long hairpin RNA, although robust and successful, is restricted to a few cell types, where long double-stranded RNA does not induce sequence-independent responses. Transgenic RNAi in mouse oocytes based on a shRNA offers several potential advantages, including simple cloning of the transgenic vector and an ability to use the same targeting construct in any cell type.

Results

Here we report our experience with shRNA-based transgenic RNAi in mouse oocytes. Despite optimal starting conditions for this experiment, we experienced several setbacks, which outweigh potential benefits of the shRNA system. First, obtaining an efficient shRNA is potentially a time-consuming and expensive task. Second, we observed that our transgene, which was based on a common commercial vector, was readily silenced in transgenic animals.

Conclusions

We conclude that, the long RNA hairpin-based RNAi is more reliable and cost-effective and we recommend it as a method-of-choice when a gene is studied selectively in the oocyte.     

Brain-targeting delivery for RNAi neuroprotection against cerebral ischemia reperfusion injury

Nanoparticles (NPs) with modification of brain-targeting molecules have been extensively exploited for therapeutic gene delivery across the blood–brain barrier (BBB). As one of the effective RNA interference (RNAi) approaches, short hairpin RNA (shRNA) has been proved to be promising in the field of gene therapy. Apoptosis signal-regulating kinase 1 (Ask1) has been reported to be an important target for gene therapy against cerebral ischemia reperfusion injury. In this study, dendrigraft poly-l-lysine (DGL) was decorated by dermorphin (a μ-opiate receptor agonist) through PEG for efficient brain-targeting, then complexed with anti-Ask1 shRNA plasmid DNA, yielding the DGL-PEG-dermorphin/shRNA NPs. The DGL-PEG-dermorphin/shRNA NPs were characterized and estimated the brain-targeting ability. In vitro, increased cellular uptake and transfection efficiency were explored; in vivo, preferable accumulation and gene transfection in brain were showed in images. The DGL-PEG-dermorphin/shRNA NPs also revealed high efficiency of neuroprotection. As a result of RNAi, corresponding mRNA was distinctly degraded, expression of Ask1 protein was obviously suppressed, apoptotic cell death was apparently decreased and cerebral infarct area was significantly reduced. Above all, DGL-PEG-dermorphin/shRNA NPs were proved to be efficient and safe for brain-targeting RNAi neuroprotection against cerebral ischemia reperfusion injury.     

慢病毒介导RNA抑制黑素瘤细胞Foxp3蛋白表达

目的:利用RNA干扰(RNAi)技术在体外干扰小鼠B16黑素瘤细胞Foxp3基因表达,探讨RNA干扰用于黑素瘤治疗的可行性。方法:针对Foxp3基因设计小干扰RNA(siR-NA),构建起短发夹状RNA(shRNA)慢病毒表达载体,并转染小鼠B16细胞,在体外诱导RNA干扰。分别采用Westernblot和RT-PCR检测Foxp3基因的表达情况;ELISA检测TGF-β1、TGF-β2和IL-10等细胞因子的变化;将干扰后的小鼠B16细胞与CD4+CD25-T淋巴细胞共培养,CCK8法检测CD4+CD25-T淋巴细胞增殖能力。结果:通过Foxp3 shRNA的转染,可实现对B16细胞Foxp3表达的沉默,可下调肿瘤细胞对CD4+CD25-T淋巴细胞增殖抑制的能力,并且下调TGF-β1、TGF-β2和IL-10等细胞因子的表达,尤其是TGF-β2的表达。结论:RNA干扰可抑制小鼠黑素瘤细胞靶基因Foxp3的表达及细胞增殖,并对CD4+CD25-T淋巴细胞增殖抑制的能力减弱,同时减弱抑制性细胞因子的分泌,为黑素瘤的基因治疗提供了新思路。     

RNAi在丙型肝炎基因治疗中的研究进展

RNA干扰（RNA interference,RNAi）技术是通过小干扰RNA(siRNA)降解具有序列同源性的mRNA分子而实现的基因表达沉默。目前将RNAi应用于丙型肝炎基因治疗的研究备受关注。siRNA能够有效抑制病毒复制和蛋白表达。但要将该技术应用于丙型肝炎临床治疗仍然存在很多问题。     

RNA干扰Skp2基因抑制T47D乳腺癌细胞生长

目的构建S期激酶相关蛋白2（skp2）RNA干扰真核细胞表达载体,研究skp2基因沉默对乳腺癌T47D细胞生长的抑制作用。方法应用Ambion公司在因特网上的设计程序设计小干扰RNA。构建3个不同的重组体转染T47D细胞,RT-PCR和Western Blotting检测skp2基因表达,细胞计数分析Skp2RNA干扰对肿瘤细胞增殖的影响。结果重组体经PCR和测序证实准确连接。转染重组体的细胞skp2的表达在mRNA和蛋白水平均明显下降。重组体转染的细胞增殖明显降低。结论用RNA干扰下调skp2表达抑制乳腺癌细胞生长,skp2可能是乳腺癌治疗的潜在靶点。     

Directing RNA interference specifically to differentiated muscle cells

A common approach for mediating RNA interference (RNAi) is to introduce DNA that encodes short hairpin RNA (shRNA), which is often contained in a plasmid that can express a shRNA in a wide variety of cell types. Muscle cells and certain other cell types grown in culture can exist in both a dividing state and in a post-mitotic, differentiated state, and it is sometimes useful to induce RNAi selectively in terminally differentiated cells to study the function of a gene, particularly when the gene is also required for propagation of dividing cells. We describe two methods for studying gene function by RNAi specifically in terminally differentiated skeletal muscle cells in culture. We developed a shRNA expression vector, based on myosin light chain 1f gene regulatory sequences, which is designed to induce shRNA expression specifically after differentiation has been initiated. We show that this vector can mediate RNAi and is only active in differentiated muscle cells. Also, we developed an adenoviral vector that is designed to be able to deliver shRNAs directly to post-mitotic muscle cells. We show that adenoviruses produced using this vector mediate RNAi in differentiated muscle cells. These methods add to the repertoire of RNAi tools that can be used for identifying genes involved in any event of interest that occurs in differentiated muscle cells.     

RNAi下调survivin表达诱导成人神经细胞瘤细胞凋亡

目的利用RNA干扰技术下调凋亡抑制因子survivin在人成神经细胞瘤细胞系SH-SY5Y中的表达,并评价其对肿瘤细胞凋亡的影响。方法将设计合成的寡核苷酸片段退火后连入pBSHH1构建survivin的短发夹RNA表达质粒pBSHH1-survivin;通过RT-PCR和Western印迹评价其对SH-SY5Y细胞内源性survivin表达的抑制作用;运用Hoechst33258核染色、DNA凝胶电泳以及AnnexinFITC染色检测survivin表达下调对SH-SY5Y细胞凋亡的影响。结果酶切和DNA测序证实survivin的短发夹RNA表达质粒构建成功;RT-PCR和Western印迹表明质粒转染SH-SY5Y细胞后能明显下调survivin的表达;Hoechst33258核染色、DNA凝胶电泳以及AnnexinⅤFITC染色显示下调survivin的表达能诱导SH-SY5Y细胞凋亡。结论成功构建了有效针对survivin的短发夹RNA表达质粒;下调survivin的表达能诱导SH-SY5Y细胞凋亡。     

siRNA体内递送策略研究进展

RNAi是由小双链RNAs触发的序列特异性转录后基因沉默,RNAi具有锁定任何引起疾病或疾病相关蛋白表达的能力,有巨大的治疗应用前景。但以治疗为目的的siRNA特异、有效的递送仍是RNAi广泛治疗应用的主要障碍。本文就目前siRNA体内递送取得的研究进展进行综述,以有助于RNAi技术安全、有效的临床应用。     

siRNA构建及转运方法研究进展

RNA干扰是一种由双链RNA引发的转录后基因沉默过程,目前已尝试将其应用于疾病治疗.但是在RNA干扰的应用研究中,最大的障碍是如何将小干扰RNA(siRNA)有效、安全地转运到靶组织和细胞.对近年来有关siRNA转运方式的研究进行综述,为寻求更好地转运方法提供可靠的依据.     

Suppression of Mamu-AG by RNA Interference

Problem  The role of placental major histocompatibility complex (MHC) class I molecules in pregnancy is not well understood. Mamu-AG, the rhesus monkey homology of human leukocyte antigen (HLA)-G expressed in the human placenta, was targeted for degradation by RNA interference (RNAi), a powerful tool to aid in determining gene function, to determine the effect that this knockdown has on NK cell function.
Method of study  A series of potential target short hairpin RNA (shRNA) sequences to suppress Mamu-AG expression was screened, which identified an optimal sequence to use in transfection experiments. Knockdown in two different Mamu-AG-expressing cell lines was measured by flow cytometry. Cytotoxicity assays were performed to correlate Mamu-AG expression with NK cell cytotoxicity.
Results  Decreased expression of Mamu-AG by short interfering RNA (siRNA) (70–80%) in cell types tested was associated with increased lysis of Mamu-AG target cells.
Conclusion  Target sequences have been identified that knocked down Mamu-AG expression by RNAi and increased lysis by NK cells. This supports the concept that NK cell receptors recognize this placental non-classical MHC class I molecule.     

DNMT1 siRNA 稳定表达载体的构建及其沉默效率的评价

目的构建能在肝癌细胞系SMMC-7721中稳定表达的高效率DNMT1的RNA干扰载体。方法合成特异性干扰DNMT1的小分子干扰RNA(small interfering RNA，siRNA)分子，并与pSUPER—GFP载体连接。脂质体转染SMMC-7721细胞，并以G418筛选稳定表达细胞系。应用逆转录-聚合酶链反应和Western印迹方法对细胞系中DNMT1的RNA表达水平和蛋白表达水平进行了分析。应用甲基化聚合酶链反应方法分析该细胞系中甲基化的基因E-钙粘着蛋白的甲基化状态。结果转染DNMT1沉默载体的SMMC-7721细胞中DNMT1的mRNA表达量为对照载体pSUPER转染SMMC-7721细胞的43％，而前者中DNMT1蛋白表达水平小于后者的10％。DNMT1的siRNA沉默效率大于90％，所构建的DNMT1的siRNA有较高的沉默效率。DNMT1的表达抑制使E-钙粘着蛋白基因启动子区出现去甲基化。结论成功构建了能稳定表达的高效率DNMT1的RNA干扰载体，但目的基因沉默后其RNA表达水平与蛋白质表达水平表现不一致，评价siRNA干扰作用时应以目的基因相应蛋白质的表达水平为准。