siRNA Dotaz Zobrazit nápovědu
Východisko. U pacientů s chronickou myeloidní leukémií jsme detekovali zvýšenou hladinu exprese genu PCNA, který by se potenciálně mohl podílet na rozvoji onemocnění. Cílem této práce bylo nalézt vhodné podmínky pro transfekci molekul PCNA-siRNA do buněčných linií K562 a MOLM-7 s up-regulovaným PCNA a zvýšenou expresi tlumit. Metody a výsledky. Klíčovými parametry úspěšného vnášení siRNA do buněk jsou typ a množství transfekčního činidla, koncentrace buněk a vnášené siRNA nebo doba inkubace transfekovaných buněk před analýzou exprese. Byla testována tato činidla: ExGene 500 (Fermentas), Metafectene (Biontex), Oligofectamine (Qiagen) a siPORT Amine (Ambion). Účinnost transfekce siRNA značených fluoresceinem byla sledována pomocí fluorescenční mikroskopie. Míra potlačení genové exprese na úrovni mRNA byla stanovována pomocí RT-PCR v reálném čase a na proteinové úrovni metodou western blotů. Jako nejvhodnější byl pro další pokusy vybrán Oligofectamine, pomocí kterého bylo dosaženo 70% poklesu hladiny mRNA genu PCNA. Dále byla zvolena 50 nM koncentrace siRNA, 1x106 buněk/ml a množství Oligofectaminu 4 µl/1ml transfekovaných buněk. Nejvhodnější doba kultivace buněk po transfekci byla 48 hodin. Závěry. Na základě výsledků této práce byl navržen protokol pro vnášení siRNA do buněčných linií K562 a MOLM-7. Tuto techniku lze přenést i na další geny potenciálně přispívající k CML a sledovat dopad siRNA-inhibice genů na expresní profil takto ovlivněných buněk. siRNA proti některým z over-exprimovaných genů by navíc mohly být v budoucnu využity pro genovou terapii CML.
Background. Increased expression of PCNA gene was detected in chronic myeloid leukemia (CML) patients in our laboratory. The gene may participate in the disease development. The aim of the study was to develop appropriate conditions for PCNA-siRNA transfection into K562 and MOLM-7 cell lines (which both have the up-regulated PCNA gene) and to silence the increased expression. Methods and Results. Key parameters of successful siRNA delivery into the cells are type and quantity of transfection reagent, cells and siRNA concentration or cultivation time before an expression analysis. Transfection reagents ExGene 500 (Fermentas), Metafectene (Biontex), Oligofectamine (Qiagen) and siPORT Amine (Ambion) were tested. Transfection efficiency was monitored by fluorescence microscopy of fluorescein labeled siRNA. Gene silencing was determined at mRNA level by real-time PCR and at protein level by western blots. As the most suitable reagent was chosen Oligofectamine, which achieved 70% decrease of PCNA mRNA level. Further, 50 nM siRNA concentration, 1x106 cells/ml and amount of Oligofectamine 4 µl per 1 ml of transfected cells were selected. The best cultivation time after siRNA delivery was 48 h. Conclusions. Based on the results of this study, transfection method for siRNA delivery into the K562 and MOLM-7 cell lines was proposed. The procedure can be transferred also on further selected genes potentially involved in CML and afterwards it will be possible to monitor the impact of siRNA-inhibition on expression profile. In the future siRNAs against some over-expressed genes would be used for gene therapy of CML.
Látky na bázi malé interferující RNA (siRNA) jsou novou třídou terapeutik určených k inhibici exprese specifických genů podílejících se na různých onemocněních. Využitím mechanismu přirozené interference RNA (RNAi) se molekuly siRNA mohou vázat na komplementární sekvence messenger RNA (mRNA), což vede k jejich degradaci a zabraňuje syntéze proteinů. Tento přístup umožňuje cílené umlčování genů, díky čemuž jsou léky siRNA zvláště slibné pro celou plejádu patologických stavů. V současné době je k dispozici 5 léčiv, která tuto technologii využívají.
Small interfering RNA (siRNA) drugs are a new class of therapeutics designed to inhibit the expression of specific genes involved in various diseases. By harnessing the natural RNA interference (RNAi) mechanism, siRNA molecules can bind to complementary messenger RNA (mRNA) sequences, leading to their degradation and preventing protein synthesis. This approach allows for targeted gene silencing, making siRNA drugs particularly promising for treating of various types of pathologic conditions. Currently we have 5 medicinal products using this technology
To assist in overcoming the inherent instability of nucleic acid-containing polyplexes in physiological solutions, we have here set out to develop removable nanocoatings for modifying the surface of siRNA-based nanoparticles. Here, N-(2-hydroxypropyl)methacrylamide (HPMA) based copolymers containing carbonylthiazolidine-2-thione (TT) reactive groups in their side chains bound via disulfide spacers to the polymeric backbone were synthesized, and these copolymers were used to coat the surface of polyplexes formed by the self-assembly of anti-Luciferase siRNA with the polycations polyethylene imine (PEI) and poly(HPMA)-grafted poly(l-lysine) (GPL). The coating process was monitored by analyzing changes in the weight-average molecular weight (M(w)), the hydrodynamic radius (R(h)), and the zeta-potential (ζ) of the polyplexes, using both static (SLS) and dynamic (DLS) light scattering methods. The outlined methods resulted in the attachment of, on average, 28 polymer molecules to the surface of the polyplexes, forming a ∼5-nm-thick hydrophilic stealth coating. Initial efforts to develop RGD-targeted coated polyplexes are also described. Atomic force microscopy (AFM) showed an angular polyplex structure and confirmed the narrow size distribution of the coated nanoparticles. The stability of the polymer-coated and uncoated polyplexes was evaluated by gel electrophoresis and by turbidity measurements, and it was found that modifying the surface of the siRNA-containing polyplexes substantially improved their stability in physiological solutions. Using polymer-coated GPL-based polyplexes containing anti-Luciferase siRNA, we finally also obtained some initial proof-of-principle for time- and concentration-dependent target-specific gene silencing, suggesting that these systems hold significant potential for further in vitro and in vivo evaluation.
INTRODUCTION: hepatocellular carcinoma (hcc) is the predominant form of primary liver cancer and the second leading cause of cancer-associated mortality worldwide. available therapies for hcc have limited efficacy due to often late diagnosis and the general resistance of hcc to anti-cancer agents; therefore, the development of novel therapeutics is urgently required. small-interfering rna (sirna) molecules are short, double-stranded rnas that specifically recognize and bind the mrna of a target gene to inhibit gene expression. despite the great therapeutic potential of sirnas towards many human tumors including hcc, their use is limited by suboptimal delivery. Areas covered: In this review, we outline the current data regarding the therapeutic potential of siRNAs in HCC and describe the development of effective siRNA delivery systems. We detail the key problems associated with siRNA delivery and discuss the possible solutions. Finally, we provide examples of the various siRNA delivery strategies that have been employed in animal models of HCC and in human patients enrolled in clinical trials. Expert opinion: Despite the existing difficulties in siRNA delivery for HCC, the increasing scientific attention and breakthrough studies in this field is facilitating the design of novel and efficient technical solutions that may soon find practical applications.
- MeSH
- buněčné linie MeSH
- hepatocelulární karcinom genetika terapie MeSH
- lidé MeSH
- malá interferující RNA aplikace a dávkování MeSH
- nádory jater genetika terapie MeSH
- protinádorové látky terapeutické užití MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
RNA interference is a powerful tool for gene silencing, which is mediated by introducing siRNA. In the present study, statistical analyses of published siRNA selection criteria, the interpretation of some criteria and systematic searching for new criteria have been carried out for CGB siRNA and siRecords databases. The results of the analyses are as follows: (i) Our study supports the two-state model of the RNA-induced silencing complex (RISC). (ii) Stable 5'-S ends of a siRNA sequence, higher stability of the whole siRNA, and low breaking energy of siRNA duplex occurs in effective siRNA sequences. Also low internal stability of the 5'-AS terminus is preferred. (iii) Secondary structure can be successfully used as an RNAi selection criterion. (iv) Several published sequence criteria have been confirmed and also new criteria have been developed. (v) Also a Target Patterns criterion, which is comparable or better than the best known criteria, has been created.
Pancreatic ductal adenocarcinoma (PDAC) is a growing medical problem associated with extensive metastasis and high mortality. Intraperitoneal (IP) administration of therapeutics promises to help the treatment of cancers originated from organs in the peritoneal cavity. In this study, we evaluated how physicochemical properties of self-assembled polycation/siRNA nanoparticles affect their IP delivery efficacy in an orthotopic PDAC model. We have examined the effect of covalent polycation modification with lipophobic and hydrophobic tetrafluoro-p-toluic acid (TFTA), hydrophobic cholesterol, and hydrophilic poly(ethylene glycol) respectively. The surface charge of the three different nanoparticles was also modulated by coating the surface with serum albumin. We found that positively charged fluorine-containing particles with lipophobic properties based on a mixture of positively charged polymeric AMD3100 CXCR4 antagonist (PAMD) and PAMD modified with TFTA (mPAMD-TFTA)/siRNA displayed the best cell uptake and transfection efficacy in vitro. Biodistribution evaluation of the nanoparticles in a syngeneic orthotopic PDAC model revealed that the fluorine-containing formulation also achieved the highest PDAC tumor accumulation after IP administration. With a combination of CXCR4 inhibition by PAMD and PLK1 downregulation by siRNA, the treatment with mPAMD-TFTA/siPLK1 showed significant inhibition of both primary and metastatic PDAC tumors. Overall, our study provides insights into and guides the design of the nanoparticles for improved IP delivery of siRNA in PDAC.
- MeSH
- halogenace * MeSH
- lidé MeSH
- malá interferující RNA MeSH
- nádorové buněčné linie MeSH
- nádory slinivky břišní * farmakoterapie MeSH
- polyelektrolyty MeSH
- tkáňová distribuce MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
