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1.
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. 相似文献
2.
Background
A variety of synthetic carriers, such as cationic polymers and lipids, have been used as nonviral carriers for small interfering RNA (siRNA) delivery. Although siRNA polyplexes and lipoplexes exhibited good gene silencing efficiencies, they often showed serious cytotoxicities, which are not useful for clinical applications. A double‐stranded RNA binding cellular protein with highly specific siRNA binding property and noncytotoxicity was used for siRNA delivery.Methods
A double‐stranded RNA binding domain (dsRBD) of human double‐stranded RNA activated protein kinase R was genetically produced and utilized to complex siRNA for intracellular delivery. For characterization of the siRNA/dsRBD complexes, decomplexation assay and RNase protection assay were performed. Cytotoxicity and target gene inhibition ability were also examined using human carcinoma cell lines.Results
The recombinantly produced polypeptide dsRBD exhibited its inherent binding activity for siRNA without sequence specificity, and the siRNA/dsRBD complexes protected siRNA from degradation by ribonucleases. Green fluorescent protein (GFP) siRNA/dsRBD complexes showed prominent down‐regulation of a target GFP gene, when an endosomal escape function was supplemented by addition of a fusogenic peptide, KALA, in the formulation.Conclusions
The results suggest that dsRBD‐based protein carriers could be successfully applied for a wide range of therapeutic siRNAs for intracellular gene inhibition without showing any cytotoxicity. Copyright © 2009 John Wiley & Sons, Ltd. 相似文献3.
4.
Small interfering RNAs (siRNA) could be the ideal inhibitor of specific gene expression if they could be delivered efficiently to their targets. Although siRNAs cannot by themselves cross cell membranes, they can enter cells with the help of transfection reagents via facilitated transport. Inside cells, however, they can still be hydrolyzed by cytoplasmic RNases before reaching their target. Here we show that poly-2'- O-(2,4-dinitrophenyl)-siRNA (DNP-siRNA) can by itself diffuse into mammalian cells and stay in the cells for several days, with no detectable degradation. Using the DNP-RNA targeting to the insulin-like growth factor receptor (IGF-IR) as an example, we demonstrate that the efficacy of this DNP-siRNA for inhibiting the growth of three different types of tumor cells is several-fold higher than that of the corresponding native siRNA. Similar results were obtained from Western blotting assay. As expected, the control siRNAs with mismatched, scrambled, and reverse sequences are all inactive. These results illustrate that both the biomembrane permeability and intracellular stability of siRNA can be greatly improved by DNP derivatization without loss of its sequence specificity. 相似文献
5.
Junsuke Hayashi Misa Nishigaki Yosuke Ochi Shun-ichi Wada Fumito Wada Osamu Nakagawa Satoshi Obika Mariko Harada-Shiba Hidehito Urata 《Bioorganic & medicinal chemistry letters》2018,28(12):2171-2174
Small interfering RNAs (siRNAs) are an active agent to induce gene silencing and they have been studied for becoming a biological and therapeutic tool. Various 2′-O-modified RNAs have been extensively studied to improve the nuclease resistance. However, the 2′-O-modified siRNA activities were often decreased by modification, since the bulky 2′-O-modifications inhibit to form a RNA-induced silencing complex (RISC). We developed novel prodrug-type 2′-O-methyldithiomethyl (MDTM) siRNA, which is converted into natural siRNA in an intracellular reducing environment. Prodrug-type 2′-O-MDTM siRNAs modified at the 5′-end side including 5′-end nucleotide and the seed region of the antisense strand exhibited much stronger gene silencing effect than non-prodrug-type 2′-O-methyl (2′-O-Me) siRNAs. Furthermore, the resistances for nuclease digestion of siRNAs were actually enhanced by 2′-O-MDTM modifications. Our results indicate that 2′-O-MDTM modifications improve the stability of siRNA in serum and they are able to be introduced at any positions of siRNA. 相似文献
6.
Tianzhi Yang Tracy Bantegui Kaitlynn Pike Raymond Bloom Roger Phipps 《Journal of liposome research》2014,24(4):270-279
One of the biggest challenges for small interfering RNAs (siRNAs) as therapeutic agents is their insufficient cellular delivery efficiency. We developed long circulating and cationic liposomes to improve the cell uptake and inhibitory effectiveness of siRNA on the expression of vascular endothelial growth factor (VEGF) in cancer cells. SiRNA liposomes were obtained by polyelectrolyte complexation between negatively charged siRNA and positively charged liposome prepared by a hydration method. Gel electrophoresis was used to evaluate the loading efficiency of siRNA on the cationic liposome. The optimized siRNA liposomes were observed to be spherical in shape and had smooth surfaces with particle sizes of 167.7?±?2.0?nm and zeta potentials of 4.03?±?0.69?mV, which had no significant change when stored at 4?°C for three months. Fluorescence-activated cell sorting studies and confocal laser scanning images indicated that the cationic liposomes significantly increased the uptake of fluorescence-labeled siRNA in cancer cells. Effects of the siRNA on the inhibition of VEGF were tested by measuring concentrations of VEGF in cell culture media via an enzyme-linked immunosorbent assay and intracellular VEGF levels using a western blotting method. The liposomal siRNA was significantly effective at inhibiting the expression of VEGF in lung, liver and breast cancer cells. Optimal liposomes could effectively deliver siRNA into cancer cells and inhibit VEGF as a therapy agent. 相似文献
7.
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。 相似文献
8.
Stanislaus A Bakhtiar A Salleh D Tiash S Fatemian T Hossain S Akaike T Chowdhury EH 《Cancer cell international》2012,12(1):30-8
Background
RNA interference (RNAi) is a powerful approach in functional genomics to selectively silence messenger mRNA (mRNA) expression and can be employed to rapidly develop potential novel drugs against a complex disease like cancer. However, naked siRNA being anionic is unable to cross the anionic cell membrane through passive diffusion and therefore, delivery of siRNA remains a major hurdle to overcome before the potential of siRNA technology can fully be exploited in cancer. pH-sensitive carbonate apatite has recently been developed as an efficient tool to deliver siRNA into the mammalian cells by virtue of its high affinity interaction with the siRNA and the desirable size distribution of the resulting siRNA-apatite complex for effective cellular endocytosis. Moreover, internalized siRNA was found to escape from the endosomes in a time-dependent manner and efficiently silence gene expression.Results
Here we show that carbonate apatite-mediated delivery of siRNA against PLC-gamma-2 (PLCG2) and calmodulin 1 (CALM1) genes has led to the sensitization of a human cervical cancer cell line to doxorubicin- and paclitaxel depending on the dosage of the individual drug whereas no such enhancement in cell death was observed with cisplatin irrespective of the dosage following intracellular delivery of the siRNAs.Conclusion
Thus, PLCG2 and CALM1 genes are two potential targets for gene knockdown in doxorubicin and paclitaxel-based chemotherapy of cervical cancer. 相似文献9.
小RNA药物应用于临床的主要技术瓶颈在于如何高效、低毒地将小RNA分子传递到它发挥功能的场所.基于细胞穿透肽在小RNA透皮给药的临床应用中所取得的进展,本文系统评述了近年来细胞穿透肽在小RNA的体内、体外传递方面的研究动态,分析了细胞穿透肽的结构改造对肽/小RNA复合物转染进入细胞发挥功能的影响,展望了细胞穿透肽作为小RNA的体内药物传递载体的发展方向. 相似文献
10.
Sulfoglucuronosyl paragloboside (SGPG), a minor glycosphingolipid of endothelial cells, is a ligand for L-selectin and has been implicated in neuro-inflammatory diseases, such as Guillian-Barré syndrome. Inflammatory cytokines, such as TNFα and IL-1β, up-regulate SGPG expression by stimulating gene expression for glucuronosyltransferases, both P and S forms (GlcATp and GlcATs), and the human natural killer antigen (HNK-1) sulfotransferase (HNK-1 ST). Transfection of a human cerebromicrovascular endothelial cell (SV-HCEC) line with HNK-1 ST siRNA down-regulated SGPG expression, inhibited cytokine-stimulated T-cell adhesion, and offered protection against apoptosis. However, the precise mechanisms of SGPG elevation in endothelial cell apoptosis and the maintenance of blood-brain or blood-nerve barrier integrity in inflammation have not been elucidated. Blocking SGPG expression inhibited cytokine-mediated stimulation of NF-κB activity but stimulated MAP kinase activity. Furthermore, elevation of SGPG by over-expression of GlcATp and GlcATs triggered endothelial cell apoptosis, with GlcATs being more potent than GlcATp. Although SGPG-mediated endothelial cell apoptosis was preceded by inhibiting the intracellular NF-κB activity, interfering with Akt and ERK activation and stimulating caspase 3 in SV-HCECs, HNK-1ST siRNA transfection also interfered with IκB phosphorylation but stimulated ERK activation. Our data indicate that SGPG is a critical regulatory molecule for maintaining endothelial cell survival and blood-brain or blood-nerve barrier function. 相似文献
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12.
The calpains play an important role in cell death and cell signalling. Caspases catalyse wholesale destruction of cellular proteins which is a major cause of cellular death. The current study looks at the function of μ‐calpain and caspase 9, using RNAi (RNA interference)‐mediated silencing, and to observe the mRNA expression level of caspase genes during satellite cell growth. The satellite cells were treated with siRNA (small interfering RNA) of μ‐calpain and caspase 9 separately. There was reduction of 16 and 24% in CAPN1 (calpain1)‐siRNA2 and CAPN1‐siRNA3 transfected cells respectively, whereas it was 60 and 56% in CAPN1‐siRNA1 and CAPN1‐siRNA4 transfected cells respectively. CAPN1‐siRNA4 and CAPN1‐siRNA1 treated cells showed more reduction in caspase 3 and 7 gene expression. CARD9 (caspase recruitment domain 9)‐siRNA1 and CARD9‐siRNA2‐treated cells showed reduction of 40 and 49% respectively. CARD9‐siRNA1 and CARD9‐siRNA2 showed an increase in caspase 3 gene expression, whereas CARD9‐siRNA2 showed reduction in caspase 7 gene expression. These results suggest a strong cross‐talk between μ‐calpain and the caspase enzyme systems. Suppression of target genes, such as μ‐calpain and caspase 9, might have genuine potential in the treatment of skeletal muscle atrophy. 相似文献
13.
Host Cell-Virus Cross Talk: Phosphorylation of a Hepatitis B Virus Envelope Protein Mediates Intracellular Signaling 总被引:6,自引:2,他引:4
Kirsten Rothmann Martina Schnlzer Gerald Radziwill Eberhard Hildt Karin Moelling Heinz Schaller 《Journal of virology》1998,72(12):10138-10147
Phosphorylation of cytosolic pre-S domains of the duck hepatitis B virus (DHBV) large envelope protein (L) was identified as a regulatory modification involved in intracellular signaling. By using biochemical and mass spectrometric analyses of phosphopeptides obtained from metabolically radiolabeled L protein, a single phosphorylation site was identified at serine 118 as part of a PX(S/T)P motif, which is strongly preferred by ERK-type mitogen-activated protein kinases (MAP kinases). ERK2 specifically phosphorylated L at serine 118 in vitro, and L phosphorylation was inhibited by a coexpressed MAP kinase-specific phosphatase. Furthermore, L phosphorylation and ERK activation were shown to be induced in parallel by various stimuli. Functional analysis with transfected cells showed that DHBV L possesses the ability to activate gene expression in trans and, by using mutations eliminating (S→A) or mimicking (S→D) serine phosphorylation, that this function correlates with L phosphorylation. These mutations had, however, no major effects on virus production in cell culture and in vivo, indicating that L phosphorylation and transactivation are not essential for hepadnavirus replication and morphogenesis. Together, these data suggest a role of the L protein in intracellular host-virus cross talk by varying the levels of pre-S phosphorylation in response to the state of the cell. 相似文献
14.
Small interfering RNA (siRNA) is a powerful tool to silence gene expression in mammalian cells including genes of viral origin. To evaluate the therapeutic efficacy of siRNA against the hepatitis B virus (HBV), we studied the effect of transfection of the HBV-inducible cell lines HepAD38 and HepAD79 with siRNA specific for the core gene of the HBV genome. HepAD38 cells produce wild-type HBV, whereas HepAD79 cells produce the lamivudine resistant YMDD variant. Transfection of HepAD38 cells with either 1.6 or 4 microg/ml siRNA resulted in a profound inhibition (72% and 98%, respectively) of viral replication (as assessed by real-time quantitative PCR). The inhibitory effect was corroborated by a marked reduction of HBV core protein synthesis in induced HepAD38 cells. In HepAD79 cells, transfected with 1.6 or 4 microg/ml HBV-specific siRNA, virus production was reduced by 75% and 89%, respectively. 相似文献
15.
ERK2 protein regulates the proliferation of human mesenchymal stem cells without affecting their mobilization and differentiation potential 总被引:1,自引:0,他引:1
Cárcamo-Orive I Tejados N Delgado J Gaztelumendi A Otaegui D Lang V Trigueros C 《Experimental cell research》2008,314(8):1777-1788
Human Mesenchymal Stem Cells (hMSC), derived mainly from adult bone marrow, are valuable models for the study of processes involved in stem cell self-renewal and differentiation. As the Extracellular signal-Regulated Kinase (ERK) signalling pathway is a major contributor to cellular growth, differentiation and survival, we have studied the functions of this kinase in hMSC activity. Ablation of ERK2 gene expression (but not ERK1) by RNA interference significantly reduced proliferation of hMSC. This reduction was due to a defect in Cyclin D1 expression and subsequent arrest in the G0/G1 phase of the cell cycle. hMSC growth is enhanced through culture medium supplementation with growth factors (GFs) such as Platelet-Derived Growth Factor (PDGF), basic Fibroblast Growth Factor (bFGF) or Epidermal Growth Factor (EGF). However, these supplements could not rescue the defect observed after ERK2 knockdown, suggesting a common signalling pathway used by these GFs for proliferation. In contrast, ERK1/2 may be dissociated from chemotactic signalling induced by the same GFs. Additionally, hMSCs were capable of differentiating into adipocytes even in the absence of either ERK1 or ERK2 proteins. Our data show that hMSCs do not require cell division to enter the adipogenic differentiation process, indicating that clonal amplification of these cells is not a critical step. However, cell-cell contact seems to be an essential requirement to be able to differentiate into mature adipocytes. 相似文献
16.
Xu Y Linde A Larsson O Thormeyer D Elmen J Wahlestedt C Liang Z 《Biochemical and biophysical research communications》2004,316(3):680-687
The concept of small interfering RNA (siRNA) has been extended to include not only short double-stranded RNA of 19-25bp, but also single-stranded antisense RNA of the same length, since such single-stranded antisense siRNAs were recently found to be able to inhibit gene expression as well. We made comprehensive comparison of double- and single-stranded siRNA functions in RNA interference (RNAi), targeting multiple sites and different mRNAs, measuring RNAi effects at different time-points and in different cell lines, and examining response curves. Duplex siRNAs were found to be more potent than single-stranded antisense siRNAs. This was verified by the observation that single-stranded antisense siRNAs, which were inefficient in some cases when used alone, could be rescued from inefficiency by sequentially transfecting with the sense siRNAs. This result suggests that the structural character of siRNA molecules might be a more important determinant of siRNA efficiency than the cellular persistence of them. 相似文献
17.
Urokinase-type plasminogen activator receptor (uPAR) is attached to cell membranes by a glycosylphosphatidylinositol (GPI) anchor, and as such is devoid of an intracellular domain, but is nevertheless able to initiate signal transduction. Herein, we report a relationship between integrins and uPAR on the surface of the human NK cell line, YT. Our data reveals that crosslinking uPAR, which mimics uPAR clustering at focal adhesion sites, causes increases in expression of the alpha(M), alpha(V), and beta(2) integrins on the surface of YT cells. Activation of the MEK/ERK signaling cascade occurs following uPAR crosslinking, as phosphorylation of both MEK 1/2 and ERK 1/2 results from receptor clustering. The MEK-specific inhibitors PD98059 and U0126 blocked MAP kinase phosphorylation; furthermore, PD98059 inhibited the increase in integrin expression induced by uPAR clustering. This study suggests that uPAR is a signaling receptor and regulator of integrins in NK cells and may impact NK cell function, including the potential for their accumulation within tumor metastases following adoptive transfer. 相似文献
18.
Sigma receptors have no known homology with other receptor systems, have no known natural ligands, but appear to play a critical role in a large diversity of cell functions. In the absence of a conventional pharmacology, siRNA technology provides a direct means of elucidating the major cell signaling pathways influenced by this receptor system. The non-transformed human lens cell line FHL124 was found to express the sigma-1 receptor (Sig-1R) and was employed for these studies. 72 h of transfection with either of the two siRNA directed against the sigma-1 receptor reduced messenger RNA and protein levels by over 70 and 60% respectively. Subsequent incubation for 96 h in culture medium (EMEM) supplemented with 5% serum gave a partial recovery of message, but there was no significant increase in protein. LDH leakage assays showed that significant cell death occurred during this time with an increased expression of caspase-3. Thrombin (10 nM) drives the growth of lens cells with a concomitant increase in ERK and Akt phosphorylation. These increases were inhibited in the cells where knockdown had occurred but not in cells exposed to scrambled siRNA. This study establishes a central role for Sig-1R in cell survival and death. 相似文献
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20.
Fengfeng Li Tao Cheng Bingfang Zeng 《Biochemical and biophysical research communications》2009,382(2):259-263
Transforming growth factor-β1 and fibroblast growth factor-2 play very important roles in fibroblast proliferation and collagen expression. These processes lead to the formation of joint adhesions through the SMAD and MAPK pathways, in which ERK2 is supposed to be crucial. Based on these assumptions, lentivirus (LV)-mediated small interfering RNAs (siRNAs) targeting ERK2 were used to suppress the proliferation and collagen expression of rat joint adhesion tissue fibroblasts (RJATFs). Among four siRNAs examined, siRNA1 caused an 84% reduction in ERK2 expression (p < 0.01) and was selected as the most efficient siRNA for use in this study. In subsequent experiments, significant downregulation of types I and III collagen were observed by quantitative RT-PCR and Western blot analyses. MTT assays and flow cytometry revealed marked inhibition of RJATF proliferation, but no apoptosis. In conclusion, LV-mediated ERK2 siRNAs may represent novel therapies or drug targets for preventing joint adhesion formation. 相似文献