Intracellular delivery of nucleic acid by cell-permeable hPP10 peptide

Although gene therapy offers hope against incurable diseases, nonreplicating transduction vectors remain lacking. We have previously characterized a cell-penetrating peptide hPP10 for the delivery of various cargoes; however, whether hPP10 can mediate nucleic acid delivery is still unknown. Here, examining via different ways, we demonstrate that hPP10 stably complexes with plasmid DNA (pDNA) and safely mediates nucleic acid transfection. hPP10 can mediate GFP-, dsRed-, and luciferase-expressing plasmids into cells with nearly the same efficiency as commercial transfection reagents Turbofectin or Lipofect. Furthermore, hPP10 can mediate Cre fusion protein delivery and pDNA transfection simultaneously in the Cre/loxp system in vitro. In addition, hPP10 fused with an RNA-binding domain can mediate delivery of small interfering RNA into cells to silence the reporter gene expression. Collectively, our results suggest that hPP10 is an option for nucleic acid delivery with efficiencies similar to that of commercial reagents.     

siRNA delivery using amphipathic cell-penetrating peptides into human hepatoma cells

Cell-penetrating peptides (CPPs) are an attractive tool for delivering membrane-impermeable compounds, including anionic biomacromolecules such as DNA and RNA, into living cells. Amphipathic helical peptides composed of hydrophobic amino acids and cationic amino acids are typical CPPs. In the current study, we designed amphipathic helical 12-mer peptides containing α,α-disubstituted α-amino acids (dAAs), which are known to stabilize peptide secondary structures. The dominant secondary structures of peptides in aqueous solution differed according to the introduced dAAs. Peptides containing hydrophobic dAAs and adopting a helical structure exhibited a good cell-penetrating ability. As an application of amphipathic helical peptides, small interfering RNA (siRNA) delivery into living human hepatoma cells was investigated. One of the peptides containing dAAs dipropylglycine formed stable complexes with siRNA at appropriate zeta-potential and size for intracellular siRNA delivery. This peptide showed effective RNA interference efficiency at short peptide length and low concentrations of peptide and siRNA. These findings will be helpful for the design of amphipathic helical CPPs as intracellular siRNA delivery.     

针对SARS冠状病毒重要蛋白的siRNA设计(英)

RNA干涉(RNA interference, RNAi)是一种特异性地导致转录后基因沉默的现象,在哺乳动物细胞中小分子干扰RNA双链体(small interfering RNA duplexes, siRNA duplexes)可以有效地诱导RNAi现象,为一些疾病的治疗开辟了新的途径.针对SARS冠状病毒(SARS coronavirus, SARS-CoV)中编码5个主要蛋白质的基因,用生物信息学的方法设计了348条候选siRNA靶标.在理论上,相应的siRNA双链体能特异地抑制SARS-CoV靶基因的表达,同时不会影响人体细胞基因的正常表达,这为进一步siRNA类药物的实验研究提供了理论基础.     

针对SARS冠状病毒重要蛋白的siRNA设计(英文)

NA干涉 (RNAinterference ,RNAi)是一种特异性地导致转录后基因沉默的现象 ,在哺乳动物细胞中小分子干扰RNA双链体 (smallinterferingRNAduplexes ,siRNAduplexes)可以有效地诱导RNAi现象 ,为一些疾病的治疗开辟了新的途径 .针对SARS冠状病毒 (SARScoronavirus ,SARS CoV)中编码 5个主要蛋白质的基因 ,用生物信息学的方法设计了3 48条候选siRNA靶标 .在理论上 ,相应的siRNA双链体能特异地抑制SARS CoV靶基因的表达 ,同时不会影响人体细胞基因的正常表达 ,这为进一步siRNA类药物的实验研究提供了理论基础     

Intelligent substance delivery into cells using cell-penetrating peptides

Cell-penetrating peptides (CPPs) are oligopeptides that can permeate the cell membrane. The use of a CPP-mediated transport system could be an excellent method for delivering cell-impermeable substances such as proteins, antibodies, antisense oligonucleotides, siRNAs, plasmids, drugs, fluorescent compounds, and nanoparticles as covalently or noncovalently conjugated cargo into cells. Nonetheless, the mechanisms through which CPPs are internalized remain unclear. Endocytosis and direct translocation through the membrane are the generally accepted routes. Internalization via both pathways can occur simultaneously, depending on cellular conditions. However, the peculiar property of CPPs has attracted many researchers, especially in drug discovery or development, who intend to deliver impermeable substances into cells through the cell membrane. The delivery of drugs using CPPs may non-invasively solve the problem of drug penetration into cells with the added benefit of low cytotoxicity. Moreover, macromolecules can also be delivered by this transport system. In this review, I discuss the possibilities and advantages of substance delivery into cells using CPPs.     

Txnip基因siRNA慢病毒表达质粒构建及促进猪前体脂肪细胞分化

硫氧还蛋白互作蛋白(thioredoxin interacting protein, Txnip)是一种氧化还原调节蛋白质,与硫氧还蛋白结合并抑制其活性,调节细胞氧化还原状态,影响细胞多种生理过程,然而其在猪脂肪细胞分化中的作用尚不明确。本文设计合成3对靶向猪Txnip基因的shRNA寡核苷酸,分别连接于重组慢病毒载体pGLV_3/H_1/GFP+Puro构建siRNA表达质粒。测序验证后,与包装质粒共转染293T细胞,获得滴度1×10~8 pfu/mL的慢病毒干扰质粒。以MOI值100转染原代培养猪前体脂肪细胞,转染率均达80%以上,其中Txnip-shRNA-2转染细胞Txnip基因沉默率达75%。转染Txnip-shRNA-2的猪前体脂肪细胞用成脂分化培养液诱导后,每隔1 d检测细胞成脂分化及相关基因表达。结果发现,其分化比阴性对照质粒转染或未转染细胞显著增强(P<0.05),PPARγ和FAS mRNA表达水平显著提高(P<0.05)。本文构建siRNA慢病毒表达质粒能有效干扰猪Txnip基因表达,Txnip表达沉默可通过上调PPARγ表达促进猪前体脂肪细胞分化。本研究提示,Txnip可能是猪脂肪细胞分化的抑制因子。     

RNA interference-mediated simultaneous silencing of four genes using cross-shaped RNA

The structural flexibility of RNA interference (RNAi)-triggering nucleic acids suggests that the design of unconventional RNAi trigger structures with novel features is possible. Here, we report a cross-shaped RNA duplex structure, termed quadruple interfering RNA (qiRNA), with multiple target gene silencing activity. qiRNA triggers the simultaneous down-regulation of four cellular target genes via an RNAi mechanism. In addition, qiRNA shows enhanced intracellular delivery and target gene silencing over conventional siRNA when complexed with jetPEI, a linear polyethyleneimine (PEI). We also show that the long antisense strand of qiRNA is incorporated intact into an RNA-induced silencing complex (RISC). This novel RNA scaffold further expands the repertoire of RNAi-triggering molecular structures and could be used in the development of therapeutics for various diseases including viral infections and cancer.     

Silencing survivin gene expression promotes apoptosis of human breast cancer cells through a caspase-independent pathway

Survivin is recognized as an attractive target in cancer therapy because of its selective overexpression in the majority of tumors. Upregulated expression of this protein correlates with increased tumor grade, recurrence risk and decreased cancer patients survival. In this study, we assessed the efficacy of two survivin-specific small interfering RNA (siRNA) constructs to inhibit T47D human breast cancer cell growth. After siRNA transfection, T47D cells showed a significant reduction in proliferation and survival exhibiting clear signs of apoptosis. pSil_1 that targeted exon 1 exhibited a stronger inhibitory effect on cell growth, and increased cell apoptosis compared to pSil_30 that targeted exon 4. Cell apoptosis was found to be mediated by translocation of the mitochondrial apoptosis inducing factor (AIF), while no changes were observed in caspase-3 activation and Bid cleavage. Thus, silencing survivin expression using siRNA strategies represents a suitable therapeutic approach to selectively modulate the survival and growth of human breast cancer cells.     

应用siRNA技术探讨MCF-7乳腺癌细胞分泌的VEGF对树突状细胞的影响

为探讨MCF-7乳腺癌细胞分泌的血管内皮生长因子（ vascular endothelial growth factor, VEGF）对树突状细胞（dendritic cell, DC）功能及其分化的影响,针对VEGF基因设计siRNA（small interfering RNA, siRNA),采用脂质体转染法以100 nmol/L最佳转染浓度导入MCF-7乳腺癌细胞（siRNA组）,以脂质体Lipofectamine　2000TM转染MCF-7 乳腺癌细胞培养上清培养正常DC作为对照（对照组）,采用ELISA法检测经siRNA 干扰VEGF基因后的MCF-7 乳腺癌细胞分泌的VEGF因子含量, Western 印迹检测VEGF蛋白表达,以探讨siRNA的基因沉默效果;以siRNA组和对照组培养上清分别培养外周血单个核细胞,用流式细胞仪检测所诱导DC表型CD1a、CD80、CD83、CD86和HLA-DR的表达,用MTT法检测转染前后两组DC 诱导的细胞毒性T淋巴细胞（cytotoxic T lymphocyte, CTL）对MCF-7细胞的细胞毒作用.结果显示,MCF-7 乳腺癌细胞培养上清能明显抑制正常DC分化成熟及抗原递呈能力,干扰VEGF基因后MCF-7 乳腺癌细胞培养上清对DC的影响明显降低,CD80、CD83、CD86和HLA-DR的表达较对照组显著升高,而CD1a表达下降（P＜0.01）.转染前后DC 诱导的CTL对MCF-7细胞的杀伤活性有明显差异（P＜0.01）.由此可见,siRNA可靶向抑制MCF-7乳腺癌细胞VEGF的表达,下调VEGF后的MCF-7 细胞上清对DC分化成熟及功能的抑制作用明显降低,从而推测VEGF在肿瘤的发生、发展和免疫抑制方面可能起着重要的作用.     

Optimized luciferase assay for cell-penetrating peptide-mediated delivery of short oligonucleotides

An improved assay for screening for the intracellular delivery efficacy of short oligonucleotides using cell-penetrating peptides is suggested. This assay is an improvement over previous assays that use luciferase reporters for cell-penetrating peptides because it has been scaled up from a 24-well format to a 96-well format and no longer relies on a luciferin reagent that has been commercially sourced. In addition, the homemade luciferin reagent is useful in multiple cell lines and in different assays that rely on altering the expression of luciferase. To establish a new protocol, the composition of the luciferin reagent was optimized for both signal strength and longevity by multiple two-factorial experiments varying the concentrations of adenosine triphosphate, luciferin, coenzyme A, and dithiothreitol. In addition, the optimal conditions with respect to cell number and time of transfection for both short interfering RNA (siRNA) and splice-correcting oligonucleotides (SCOs) are established. Optimal transfection of siRNA and SCOs was achieved using the reverse transfection method where the oligonucleotide complexes are already present in the wells before the cells are plated. Z′ scores were 0.73 for the siRNA assay and 0.71 for the SCO assay, indicating that both assays are suitable for high-throughput screening.     

Induction of caspase-8 in human cells by the extracellular administration of peptides containing a C-terminal SLV sequence

Summary Extracellular administration of a membrane permeable model peptide containing the tripeptide sequence, SLV, at the C-terminus to human endothelial and kidney cells resulted in an induction of caspase-8 (FLICE), the apical enzyme of the apoptosis caseade. The unmodified or N-terminally SLV-tagged peptide had no effect, thereby eliminating an unspecific induction of apoptosis as the cause of the caspase activity observed. Drastic alterations of primary structure and structure forming properties of the carrier peptide did not significantly influence the caspase-8 inducing activity of the C-terminal SLV-tag, supporting previous findings that translocation into the cell interior is a more general ability of peptides.     

Induction of caspase-8 in human cells by the extracellular administration of peptides containing a C-terminal SLV sequence

Extracellular administration of a membranepermeable model peptide containing the tripeptide sequence,SLV, at the C-terminus to human endothelial and kidney cellsresulted in an induction of caspase-8 (FLICE), the apicalenzyme of the apoptosis cascade. The unmodified or N-terminally SLV-tagged peptide had no effect, therebyeliminating an unspecific induction of apoptosis as the causeof the caspase activity observed. Drastic alterations ofprimary structure and structure forming properties of thecarrier peptide did not significantly influence the caspase-8inducing activity of the C-terminal SLV-tag, supportingprevious findings that translocation into the cell interior isa more general ability of peptides.     

The Voltage-dependent Anion Channel 1 Mediates Amyloid β Toxicity and Represents a Potential Target for Alzheimer Disease Therapy

The voltage-dependent anion channel 1 (VDAC1), found in the mitochondrial outer membrane, forms the main interface between mitochondrial and cellular metabolisms, mediates the passage of a variety of molecules across the mitochondrial outer membrane, and is central to mitochondria-mediated apoptosis. VDAC1 is overexpressed in post-mortem brains of Alzheimer disease (AD) patients. The development and progress of AD are associated with mitochondrial dysfunction resulting from the cytotoxic effects of accumulated amyloid β (Aβ). In this study we demonstrate the involvement of VDAC1 and a VDAC1 N-terminal peptide (VDAC1-N-Ter) in Aβ cell penetration and cell death induction. Aβ directly interacted with VDAC1 and VDAC1-N-Ter, as monitored by VDAC1 channel conductance, surface plasmon resonance, and microscale thermophoresis. Preincubated Aβ interacted with bilayer-reconstituted VDAC1 and increased its conductance ∼2-fold. Incubation of cells with Aβ resulted in mitochondria-mediated apoptotic cell death. However, the presence of non-cell-penetrating VDAC1-N-Ter peptide prevented Aβ cellular entry and Aβ-induced mitochondria-mediated apoptosis. Likewise, silencing VDAC1 expression by specific siRNA prevented Aβ entry into the cytosol as well as Aβ-induced toxicity. Finally, the mode of Aβ-mediated action involves detachment of mitochondria-bound hexokinase, induction of VDAC1 oligomerization, and cytochrome c release, a sequence of events leading to apoptosis. As such, we suggest that Aβ-mediated toxicity involves mitochondrial and plasma membrane VDAC1, leading to mitochondrial dysfunction and apoptosis induction. The VDAC1-N-Ter peptide targeting Aβ cytotoxicity is thus a potential new therapeutic strategy for AD treatment.     

Synthesis,characterization, and biological activity of poly(arginine)‐derived cancer‐targeting peptides in HepG2 liver cancer cells

The solid‐phase synthesis, structural characterization, and biological evaluation of a small library of cancer‐targeting peptides have been determined in HepG2 hepatoblastoma cells. These peptides are based on the highly specific Pep42 motif, which has been shown to target the glucose‐regulated protein 78 receptors overexpressed and exclusively localized on the cell surface of tumors. In this study, Pep42 was designed to contain varying lengths (3–12) of poly(arginine) sequences to assess their influence on peptide structure and biology. Peptides were effectively synthesized by 9‐fluorenylmethoxycarbonyl‐based solid‐phase peptide synthesis, in which the use of a poly(ethylene glycol) resin provided good yields (14–46%) and crude purities >95% as analyzed by liquid chromatography–mass spectrometry. Peptide structure and biophysical properties were investigated using circular dichroism spectroscopy. Interestingly, peptides displayed secondary structures that were contingent on solvent and length of the poly(arginine) sequences. Peptides exhibited helical and turn conformations, while retaining significant thermal stability. Structure–activity relationship studies conducted by flow cytometry and confocal microscopy revealed that the poly(arginine) derived Pep42 sequences maintained glucose‐regulated protein 78 binding on HepG2 cells while exhibiting cell translocation activity that was contingent on the length of the poly(arginine) strand. In single dose (0.15 mM) and dose‐response (0–1.5 mM) cell viability assays, peptides were found to be nontoxic in human HepG2 liver cancer cells, illustrating their potential as safe cancer‐targeting delivery agents. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Engineering responsiveness to cell culture stresses: growth arrest and DNA damage gene 153 (GADD153) and the unfolded protein response (UPR) in NS0 myeloma cells

The successful movement of a newly synthesized protein through the endoplasmic reticulum (ER) and associated membranous compartments is dependent on appropriate recognition by complex processing systems. Failure to perceive appropriately processed or modified intermediates in the pathway can initiate a series of cellular signaling events (ER stress or unfolded protein response, UPR) that can lead to cell apoptosis and loss of biomass in culture processes. We have shown that expression of growth arrest and DNA damage gene 153 (GADD153) is associated with recognition of damaged or mis-processed proteins within the secretory processes of CHO and NS0 myeloma cells. To directly characterize the roles of GADD153 in UPR-directed apoptosis, we have generated stable clones of NS0 myeloma cells with elevated (constitutive and inducible) and deleted GADD153 expression. Although GADD153 is a robust indicator of the onset of ER stress or the UPR, GADD153 expression alone is not sufficient to provoke NS0 myeloma apoptosis and it is not required for apoptosis to occur.     

Chemically modified siRNA prolonged RNA interference in renal disease

BACKGROUND: We previously demonstrated that transfection of synthetic short interfering RNAs (siRNAs) targeting against TGF-beta1 could be effective and therapeutic in silencing TGF-beta1 expression in glomerulus, thereby ameliorated the progression of matrix expansion in anti-Thy-1 model of glomerulonephritis. However, a major concern in applying RNAi to gene therapy is the prolonged existence of silencing potential in vivo. METHOD: We examined the duration of siRNA stability in kidney and muscle, and checked the tissue distribution of siRNase, eri-1. Thereafter, we tested the effect of chemically modified siRNA called siSTABLE on progressive glomerulosclerosis model. RESULTS: A single introduction of siRNA for EGFP (siEGFP) or its expression vector into kidney resulted in the reduction of masangial EGFP expression only for up to two weeks, while transfection of siEGFP into the pretibial muscle silenced EGFP expression unexpectedly for more than 90 days. These observations could be explained by the different expression of eri-1 between kidney and muscle. In addition, transfection of ERI-1-resistant siSTABLE for TGF-beta1 significantly reduced glomerular matrix deposition in progressive glomerulosclerosis model.Conclusion: Treatment with siRNA resistant to eri-1 may be effective and promising strategy for progressive renal disease.     

假基因鉴定及其功能分析

被称为"垃圾基因"的假基因是真核生物基因组中的重要组成部分。近年来对假基因的功能研究表明其并非是基因组中的沉默成员。如一些假基因参与RNA转录,一些假基因转录本能够形成小干扰RNA(siRNA),通过小RNA干扰作用调节功能基因。另外,还有研究发现,一些假基因能够通过microRNA调节肿瘤抑制因子。然而,对假基因功能的深入挖掘需要建立在对其更精准、更全面的鉴定基础之上。随着各物种全基因组测序的完成及序列比对算法的完善,全面而又精确地鉴定假基因已经成为可能。下文就近年来假基因相关鉴定方法、调节功能以及在进化上的意义进行了阐述,并对未来假基因研究方向进行了展望。     

Arginine-rich cell-penetrating peptides deliver gene into living human cells

Transgenesis is a process that introduces exogenous nucleic acids into the genome of an organism to produce desired traits or evaluate function. Improvements of transgenic technologies are always important pursuit in the last decades. Recently, cell-penetrating peptides (CPPs) were studied as shuttles that can internalize into cells directly and serve as carriers to deliver different cargoes into cells. In the present study, we evaluate whether arginine-rich CPPs can be used for gene delivery into human cells in a noncovalent fashion. We demonstrate that three arginine-rich CPPs (SR9, HR9, and PR9) are able to transport plasmid DNA into human A549 cells. For the functional gene assay, the CPP-delivered plasmid DNA containing the enhanced green fluorescent protein (EGFP) coding sequence could be actively expressed in cells. The treatment of calcium chloride did not facilitate the CPP-mediated transfection efficiency, but enhance the gene expression intensity. Mechanistic studies further revealed that HR9/DNA complexes mediate the direct membrane translocation pathway for gene delivery. Our results suggest that arginine-rich CPPs, especially HR9, appear to be a high efficient and promising tool for gene transfer.