siRNA Concentration Research Articles

Antiviral Activity of Specific siRNA against Hemagglutinin and Neuraminidase of Influenza Virus H1N1 in MDCK Cell Culture

H1N1 influenza virus causes respiratory syndrome on the upper respiratory system. Hemagglutinin (HA) and Neuraminidase (NA) proteins are the most important viral proteins that play a role in attachment and facilitates of releasing virus respectively. Short interfering RNA (siRNA) plays notable a roles in the RNA interference (RNAi) pathway and interferes the expression of specific genes with complementary nucleotide sequences by breaking down mRNA function and stopping transcription and translation. To explore shutting down HA and NA, siRNA were designed and genes of H1N1 are inhibited by siRNA molecules in MDCK cell culture. The cells were infected with virus and then treated by different concentration of siRNA. The HA, NA gene expression and the viral load were investigated by Real Time PCR between 24 to 72 hours. The cells were infected with H1N1 virus at a multiplicity of infection (MOI: 3) and then treated with different concentration of siRNA (0.5-6 nmol/L) and monitored for 72 hours post transfection. Interestingly, the viral load extremely decreased at first hours, even the viral load was not detectable after 48 hours. Three nmol/L concentration of siRNA for HA was more effective. In case of siRNA for NA, 3 nmol/L concentrations shows efficacy and also cytopathic effect (CPE) was not observed in 3 nmol/L concentrations in MDCK cells. This study suggests that siRNA against NA and HA, silenced viral genes in H1N1 influenza virus infected cells. Results showed that siRNA is an efficient tool for silencing influenza virus infection. The advantage of siRNA is the specific inhibition of mRNA which can be used for inhibition of other flu viral genes and/or other viruses.

Multiple siRNA delivery against cell cycle and anti-apoptosis proteins in breast cancer and normal cells

Event Abstract Back to Event Multiple siRNA delivery against cell cycle and anti-apoptosis proteins in breast cancer and normal cells Manoj Parmar1*, Bárbara E. Arteaga Ballesteros2*, Timothy Fu2*, Remant Bahadur Kc2*, Hamidreza Montazeri Aliabadi3*, Judith C. Hugh4* and Hasan Uludag1, 2, 5* 1 University of Alberta, Faculty of Pharmacy and Pharmaceutical Sciences, Canada 2 University of Alberta, Department of Chemical and Materials Engineering, Canada 3 Chapman University, School of Pharmacy, United States 4 University of Alberta, Department of Laboratory Medicine and Pathology, Canada 5 University of Alberta, Department of Biomedical Engineering, Canada Introduction: Current therapies for breast cancer induce severe side effects that warrant a search for alternative therapies. Short-interfering RNA (siRNA)-based therapeutics directly target and silence specific genes critical for cancer cells, thus holding promise as an effective therapy with less side effects. Given the unregulated cell cycle progression in cancer cells, proteins involved in cell cycle regulation such as spindle assembly checkpoint kinase TTK and cell-division cycle protein 20 (CDC20) are promising targets to stop uncontrolled cell growth. Other targets that might act in conjunction with cell cycle proteins are anti-apoptosis proteins such as survivin [1], which are often up-regulated in transformed cells. We hypothesize that the dual silencing of cell cycle and anti-apoptosis proteins might be exceptionally effective and specific for treatment of breast cancer. Here, we explored the combinational siRNA therapy in breast cancer as well as effects on normal cells in vitro. Methods & Results: Since siRNA is highly labile and anionic, we synthesized a library of cationic and hydrophobic (lipid-modified) polymers from 0.6, 1.2 and 2.0 kDa polyethylenimines (PEI). We screened this library for delivery of CDC20 siRNA in MDA-MB-231 cells, where 1.2 kDa PEI substituted with linoleic acid (PEI-LA) was found to be the most effective [2,][3]. We additionally determined efficacy of 6 siRNAs targeting at different locations of TTK gene; all TTK siRNAs inhibited growth of MDA-MB-231 and MCF7 breast cancer cells (as assessed by MTT Assay), which was further confirmed with the down-regulation of TTK transcripts by qPCR. We then explored the feasibility of combinational siRNA therapy by mixing specific siRNAs with polymers at different weight ratios to form complexes. TTK/CDC20 and CDC20/Survivin siRNA combinations decreased the growth of MDA-MB-231 cells significantly, while only TTK/CDC20 combination inhibited MCF7 cell growth significantly at a relatively low (15 nM each) siRNA concentration (Figure 1). The transcripts of each of these targets were significantly down-regulated in specific siRNA treated cells compared to scrambled siRNA treated cells. The combinational siRNA delivery was also performed in normal breast epithelial MCF10A cells, human umbilical vein endothelial cells (HUVEC) and human bone marrow stromal cells (HBMSC) to determine the side effects of siRNA treatments using 15 nM each siRNA at different siRNA:polymer ratios. The combinational siRNA therapy at lower ratio (1:2) showed higher side effects of siRNA treatment in these cells compared to higher ratios (1:4, 1:8; Figure 2). Based on relative effect of combinational siRNAs in breast cancer and normal cells, TTK/CDC20 siRNA combination inhibited MDA-MB-231 (33%) and MCF7 (24%) cell growth at 1:4 siRNA:polymer ratio without showing much side effects in MCF10A and HUVEC cells (Figure 2D). However, this combination showed significant side effects in HBMSC cells (33%). Conclusions: This study has indicated improved efficacy of combinational siRNA therapy against cell cycle and anti-apoptosis proteins in breast cancer therapy. The lipid-substituted polymers could serve as a viable platform for delivery of multiple siRNAs against critical targets. However, the siRNA therapy has also showed side effects on non-malignant (normal) cells in vitro and more selective therapies might be needed to target cancer cells solely. Manoj Parmar is a recipient of Women and Children's Health Research Institute (WCHRI) Graduate Studentship Grant and Alberta Innovates: Health Solutions (AIHS) Graduate Studentship.; This study was supported by operating grants from Canadian Breast Cancer Foundation (CBCF) and Natural Sciences and Engineering Council of Canada (NSERC).

Mesoporous nanocarriers with internal cavity for the delivery of siRNA for bone regeneration

Event Abstract Back to Event Mesoporous nanocarriers with internal cavity for the delivery of siRNA for bone regeneration Jungju Kim1, 2, Tsendmaa Bold1, 2, Jeong-Hyun Ryu1, 2, Roman Perez1, 2 and Hae-Won Kim1, 2, 3 1 dankook university, Tissue Regeneration Engineering, Korea 2 dankook university, Nanobiomedical Science & BK21 PLUS NBM Global Research Center for Regenerative Medicine, Korea 3 dankook university, Biomaterials Science, College of Dentistry, Korea Introduction: Nanocarriers have great interest in the field of tissue engineering for the delivery of drugs and genes. Among other issues, the loading of those molecules in sufficient amount and the sustained release are considered of special importance for therapeutic efficacy of the delivery system. For this purpose, we developed a mesoporous silica nanocarrier with an internal cavity to allow high loading capacity and sustained delivery of small interference RNA (siRNA). Here, we delivered siRNA to downregulate Plekho-1 gene, which has been shown to significantly enhance bone regeneration. We investigated the ability to load and release the siRNA as well as the cellular interaction and osteogenic potential. Materials and Methods: Apatite nanorods were used as sacrifice core templates which were then sheathed with mesoporous silica through a sol-gel reaction. Apatite was then selectively removed by an acid treatment. The surface of the nanocarriers was aminated with APTES for proper siRNA loading. The nanocarriers were characterized by transmission electron microscopy (TEM), energy-dispersive X-ray spectroscopy (EDX), Fourier transform infrared spectroscopy (FT-IR), and Brunauer Emmett Teller (BET). The loading and delivery of the siRNA was quantified in the range of 10 to 100 µg/ml. In order to observe cellular interactions, pre-osteoblastic cells were cultured with the siRNA-loaded nanocarriers at the different siRNA concentrations. The cell viability, intracellular uptake capacity, gene silencing efficiency, and bone-related gene expressions were investigated. Results and Discussion: Mesoporous and internally-hollowed silica nanocarriers were produced well, as observed by TEM images, which presenting an internal cavity of ~20 nm. Compared to the dense nanocarriers, the surfaces area and pore volume of developed nanocarriers. Due to the enhanced surface area and pore volume, the siRNA loading capacity increased 5-fold. The unique pore morphology and characteristics allowed sustained release of the siRNA from the developed nanocarriers for up to 18 days, which was significantly different from the dense nanocarriers which completely released the cargo after only 3 days. Cellular viability of the developed nanocarriers was high for the studied concentrations, allowing therefore high doses of the nanocarriers and the loading of siRNA to be delivered. The siRNA-loaded nanocarriers were successfully taken up by the pre-osteoblastic cells, with a cellular uptake ~90%, and silenced the expression of the target gene by 80%, which subsequently induced an upregulation of osteoblasts-related genes. Conclusion: Mesoporous nanocarriers with an internal cavity showed the capacity of high loading and sustained release of siRNA molecules. The siRNA-delivering nanocarriers successfully downregulated Plekho-1 target genes while upregulating osteogenic genes in vitro. Further in vivo study remains to investigate the practical efficacy of the nanocarriers for bone regeneration.

Cyp51A gene silencing using RNA interference in azole‐resistant Aspergillus fumigatus

An increasing number of reports have described the emergence of acquired resistance of Aspergillus fumigatus to azole compounds. The primary mechanism of resistance in clinical isolates is the mutation of the azole drug target enzyme, which is encoded by the cyp51A gene. The aim of this study was to evaluate the impact of silencing the cyp51A gene in azole-resistant A. fumigatus isolates. A 21-nucleotide small-interfering RNA (siRNA) was designed based on the cDNA sequence of the A. fumigatus cyp51A gene. After silencing the cyp51A gene in germinated conidia (15, 20, 25 and 50 nM), azole-resistant A. fumigatus was cultured on broth media and gene expression was analysed by measuring the cyp51A mRNA level using RT-PCR assay. Hyphae were successfully transfected by siRNA and expression of the cyp51A gene was significantly reduced by siRNA at the concentration of 50 nM (P ≤ 0.05). In addition, at this siRNA concentration, the minimum inhibitory concentration of itraconazole for the treated cells was decreased, compared with that for untreated control cells, from 16 to 4 μg/ml.

Receptor‐targeted liposome‐peptide‐siRNA nanoparticles represent a novel and efficient siRNA delivery system to prevent conjunctival fibrosis

PurposeGlaucoma is the leading cause of irreversible blindness worldwide and fibrosis is the main cause of failure of glaucoma surgery. We have previously described how the Myocardin‐related transcription factor/Serum response factor (MRTF/SRF) pathway is intricately linked to all the key pathways in ocular fibrosis. Our aim was to develop a novel liposome‐peptide‐siRNA (LYR) nanoparticle as an efficient delivery system for MRTF siRNA in conjunctival fibrosis.MethodsThe LYR nanoparticles were characterised with regard to particle size and zeta potential. Real‐time qPCR and western blotting were used to compare the silencing efficiency in human Tenon's fibroblasts using different MRTF siRNA concentrations, targeting peptides, and liposomes. The cytotoxicity of the LYR nanoparticles was assessed using the MTT cell assay. Three‐dimensional fibroblast‐populated collagen matrices were also used as a functional assay to measure contraction in vitro.ResultsAll LYR nanoparticles were strongly cationic with sizes around 100 nm and PDIs < 0.4. The LYR nanoparticles efficiently silenced the MRTF gene by 76% and 84% using 50 nM and 100 nM siRNA respectively. The MRTF gene was also efficiently silenced by 76% and 75% using the targeting peptides Y and ME27 respectively. The MRTF protein expression was significantly decreased by the LYR nanoparticles. The non‐PEGylated liposome formulations showed higher silencing efficiency than the cationic PEGylated formulations. The MRTF nanoparticles were also not cytotoxic at 50 nM siRNA concentration and prevented matrix contraction after a single transfection treatment.ConclusionsThis is the first study to show that receptor‐targeted liposome‐peptide‐siRNA nanoparticles represent an efficient and safe siRNA delivery system that could be used to prevent fibrosis after glaucoma surgery.

Receptor‐targeted liposome‐peptide‐siRNA nanoparticles represent a novel and efficient siRNA delivery system to prevent conjunctival fibrosis

PurposeGlaucoma is the leading cause of irreversible blindness worldwide and fibrosis is the main cause of failure of glaucoma surgery. We have previously described how the Myocardin‐related transcription factor/Serum response factor (MRTF/SRF) pathway is intricately linked to all the key pathways in ocular fibrosis. Our aim was to develop a novel liposome‐peptide‐siRNA (LYR) nanoparticle as an efficient delivery system for MRTF siRNA in conjunctival fibrosis.MethodsThe LYR nanoparticles were characterised with regard to particle size and zeta potential. Real‐time qPCR and western blotting were used to compare the silencing efficiency in human Tenon's fibroblasts using different MRTF siRNA concentrations, targeting peptides, and liposomes. The cytotoxicity of the LYR nanoparticles was assessed using the MTT cell assay. Three‐dimensional fibroblast‐populated collagen matrices were also used as a functional assay to measure contraction in vitro.ResultsAll LYR nanoparticles were strongly cationic with sizes around 100 nm and PDIs < 0.4. The LYR nanoparticles efficiently silenced the MRTF gene by 76% and 84% using 50 nM and 100 nM siRNA respectively. The MRTF gene was also efficiently silenced by 76% and 75% using the targeting peptides Y and ME27 respectively. The MRTF protein expression was significantly decreased by the LYR nanoparticles. The non‐PEGylated liposome formulations showed higher silencing efficiency than the cationic PEGylated formulations. The MRTF nanoparticles were also not cytotoxic at 50 nM siRNA concentration and prevented matrix contraction after a single transfection treatment.ConclusionsThis is the first study to show that receptor‐targeted liposome‐peptide‐siRNA nanoparticles represent an efficient and safe siRNA delivery system that could be used to prevent fibrosis after glaucoma surgery.

Target gene delivery from targeting ligand conjugated chitosan–PEI copolymer for cancer therapy

In this study, we designed a novel carrier which was having low cytotoxicity, site-specific target function, and high transfection efficiency using low molecular weight water soluble O-carboxymethyl chitosan (OCMCh), branched low molecular weight poly(ethyleneimine) (bPEI), and targeting ligand (epitope type, HER-2/neu). OCMCh/bPEI/targeting ligand, HPOCP copolymer, and targeting ligand-modified polyamphoteric polymer, and were prepared by chemical reaction and characterized by 1H NMR and FT-IR. The binding affinity, protecting efficiency, and releasing ability of gene/HPOCP polyplex were confirmed by gel retardation assay. The pDNA(pEGFP)/HPOCP polyplexes showed high gene transfection efficiency in HCT 119 cell. In addition, siRNA/HPOCP polyplexes formed spherical shape and have particle sizes from 100 to 300nm. The siRNA/HPOCP polyplexes have lower cytotoxicity than PEI in the all of siRNA concentrations ranging from 0 to 2μg/μL in HEK 293 cells. The cell viability of siRNA/HPOCP polyplexes was performed in SK-Br3 cells with VEGF siRNA or BCL2 siRNA. In addition, confocal laser-scanning microscopy and flow cytometry assay were performed for cellular localization and cellular uptake efficiency of siRNA/HPOCP polyplexes. The results of the present study demonstrate that HPOCP copolymer is a good candidate as gene delivery carriers for gene delivery system or gene therapy.

Intracellular siRNA delivery dynamics of integrin-targeted, PEGylated chitosan–poly(ethylene imine) hybrid nanoparticles: A mechanistic insight

Integrin-targeted nanoparticles are promising for the delivery of small interfering RNA (siRNA) to tumor cells or tumor endothelium in cancer therapy aiming at silencing genes essential for tumor growth. However, during the process of optimizing and realizing their full potential, it is pertinent to gain a basic mechanistic understanding of the bottlenecks existing for nanoparticle-mediated intracellular delivery. We designed αvβ3 integrin-targeted nanoparticles by coupling arginine–glycine–aspartate (RGD) or RGD peptidomimetic (RGDp) ligands to the surface of poly(ethylene glycol) (PEG) grafted chitosan–poly(ethylene imine) hybrid nanoparticles. The amount of intracellular siRNA delivered by αvβ3-targeted versus non-targeted nanoparticles was quantified in the human non-small cell lung carcinoma cell line H1299 expressing enhanced green fluorescent protein (EGFP) using a stem-loop reverse transcription quantitative polymerase chain reaction (RT-qPCR) approach. Data demonstrated that the internalization of αvβ3-targeted nanoparticles was highly dependent on the surface concentration of the ligand. Above a certain threshold concentration, the use of targeted nanoparticles provided a two-fold increase in the number of siRNA copies/cell, subsequently resulting in as much as 90% silencing of EGFP at well-tolerated carrier concentrations. In contrast, non-targeted nanoparticles mediated low levels of gene silencing, despite relatively high intracellular siRNA concentrations, indicating that these nanoparticles might end up in late endosomes or lysosomes without releasing their cargo to the cell cytoplasm. Thus, the silencing efficiency of the chitosan-based nanoparticles is strongly dependent on the uptake and the intracellular trafficking in H1299 EGFP cells, which is critical information towards a more complete understanding of the delivery mechanism that can facilitate the future design of efficient siRNA delivery systems.

肝細胞癌細胞株MHCC97Hのサバイビン発現,増殖およびアポトーシスに及ぼす低分子干渉RNAの効果【Powered by NICT】

Objective To observe the effect of small interfering RNA(siRNA)targeting Survivin gene on urviving expression, proliferation and apoptosis of hepatocellular carcinoma cell line MHCC-97H. Methods Survivin sequence specific siRNA was designed and synthesized.siRNA/liposome complex was transfected into hepatocellular carcinoma cell line MHCC- 97H.The MHCC-97H cells were divided into Survivin siRNA group(Si-survivin), negative control siRNA group(NC group)and blank group(normal control group). Survivin mRNA and protein expressions were detected by reverse transcription-PCR and Western blot, respectively.The proliferation of MHCC-97H was measured by methythiazolydiphenyl-tetrazolium bromide assay.The Annexin V/PI double labeled flow cytometry was employed to measure the apoptosis at 24 h after transfection in different concentrations of Survivin siRNA(12.5, 25.0 and 50.0 nmol/L, respectively). Results After 48 h of transfection, the Survivin mRNA levels were 0.55±0.16(Si-survivin group), 0.85±0.28(NC group)and 0.93±0.40(normal control group), respectively, which were significantly different among 3 groups(F=414, P<0.01). The level of Survivin mRNA was the lowest in Si-survivin group, which was statistically different with NC group and normal control group(t=-20.56, -28.37, all P<0.001). The levels of Survivin protein expression in 3 groups were 0.602±0.005 (Si-survivin group), 0.835±0.007(NC group)and 0.993±0.003(normal control group)at 48 h after transfection, which were statistically different among 3 groups(F=238, P<0.01). The lowest level of protein expression was in Si-survivin group, which was statistically different with NC group and normal control group(t=-40.17, -66.03, all P<0.001). After 72 h and 96 h of transfection, the inhibitory rate of cell growth was significantly higher in Si-survivin group[(19.5±3.6)%, (12.0±0.9)%] compared with that in NC group[(3.6±0.9)%, (-1.3±6.1)%](t=36.18, 42.53, all P<0.05). The apoptosis rates in 12.5, 25.0, 50.0 nmol/L Survivin siRNA were (22.64±2.54)%, (35.37±3.28)% and (53.28±4.35)%, respectively.However, in NC group and normal control group, the apoptosis rates were (8.77±1.25)% and (9.72±1.37)%.The rates were statistically different among those 5 groups(F=35.93, P<0.01). And in the apoptosis rates of siRNA groups in different concentrations were statistically different when compared between each two groups(t=-29.73, -38.57, all P<0.001). Conclusion Survivin specific siRNA can inhibit the proliferation and induce the apoptosis by blocking Survivin gene expression in hepatocellular carcinoma cell line MHCC-97H. Key words: Hepatocellular carcinoma; Survivin; Small interfering RNA

Bio-inspired pulmonary surfactant-modified nanogels: A promising siRNA delivery system

Inhalation therapy with small interfering RNA (siRNA) is a promising approach in the treatment of pulmonary disorders. However, clinical translation is severely limited by the lack of suitable delivery platforms. In this study, we aim to address this limitation by designing a novel bioinspired hybrid nanoparticle with a core–shell nanoarchitecture, consisting of a siRNA-loaded dextran nanogel (siNG) core and a pulmonary surfactant (Curosurf®) outer shell. The decoration of siNGs with a surfactant shell enhances the colloidal stability and prevents siRNA release in the presence of competing polyanions, which are abundantly present in biofluids. Additionally, the impact of the surfactant shell on the biological efficacy of the siNGs is determined in lung cancer cells. The presence of the surfactants substantially reduces the cellular uptake of siNGs. Remarkably, the lowered intracellular dose does not impede the gene silencing effect, suggesting a crucial role of the pulmonary surfactant in the intracellular processing of the nanoparticles. In order to surmount the observed reduction in cellular dose, folate is incorporated as a targeting ligand in the pulmonary surfactant shell to incite receptor-mediated endocytosis. The latter substantially enhances both cellular uptake and gene silencing potential, achieving efficient knockdown at siRNA concentrations in the low nanomolar range.

Biodegradable stearylated peptide with internal disulfide bonds for efficient delivery of siRNA in vitro and in vivo.

RNA-based delivery system for cancer therapy remains a challenge. In this study, a stearyl-peptide (SHR) was synthesized using arginine, histidine, cysteine, and stearyl moieties. Further, the stearyl-peptides were cross-linked by disulfide bonds to obtain cross-linked polypeptides (SHRss) with different molecular weight (SHRss1, SHRss2, SHRss3, SHRss4). The SHRss could effectively condense small interfering RNA (siRNA) into polyplexes with a hydrodynamic size of 100-300 nm and zeta potential of 20-40 mV. Flow cytometry and confocal laser scanning microscope studies revealed high cellular uptake and rapid dissociation behavior of SHRss2/siRNA complexes. Long-lasting high concentration of siRNA in cytoplasm was observed even at 24 h after SHRss2/Cy3-siRNA transfection. Compared with SHR, the SHRss showed much improved siRNA interference efficiency targeting luciferase on Luc-Hela cells. Moreover, SHRss2 exhibited higher interference efficiency and slower decay rate on Luc-Hela cells than Lipofectamine 2000 and SHR. In addition, much weaker expression of red fluorescence protein was also observed on SHRss2/simCh-treated mCherry-HEK293 cells than Lipofectamine 2000 and SHR. The SHRss did not induce cytotoxicity at siRNA concentrations of 25-200 nM under transfection. The in vivo studies demonstrated the gene interference efficiency of SHRss2/siRNA complexes. Our studies indicated that the SHRss are promising and efficient nonviral vectors for siRNA delivery.

Abstract A57: Effects of pre-exposure to siRNA on silencing response: Do cells become resistant to siRNA silencing?

Abstract Development of resistance against cytotoxic effect of different generations of anticancer drugs is considered inevitable and remains a major concern for cancer therapy. An alternative therapeutic approach based on protein silencing via RNA interference (RNAi) have shown considerable promise; however, our information about the factors affecting siRNA efficiency and the limitations to their efficacy are limited. This study focused on determining the possibility of development of resistance against siRNA treatment as a result of repeated exposure to siRNA that could affect cellular internalization of the particles, silencing efficiency at mRNA level, and ultimately, the observed cellular response. Two approaches to siRNA exposure were undertaken: one involving a single high concentration exposure to eradicate siRNA-responsive cells, and one involving multiple exposures with a gradually increasing siRNA concentration to study the potential intracellular adaptations. Our results demonstrated a temporary decrease in silencing efficiency at mRNA level without significant alteration in siRNA cellular uptake. We conclude that cells would respond to repeated siRNA exposure in a similar fashion after a temporary initial alteration; however, we could not rule out the possibility of a change in phenotypical response (e.g., number of viable cells) as a result of adjustments in (or selection of cells reliant on) alternative pathways. Citation Format: Hamidreza Montazeri Aliabadi, Parvin Mahdipoor, Cezary Kucharsky, Nicole Chan, Hasan Uludag. Effects of pre-exposure to siRNA on silencing response: Do cells become resistant to siRNA silencing? [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr A57.

Long (27-nucleotides) small inhibitory RNAs targeting E6 protein eradicate effectively the cervical cancer cells harboring human papilloma virus.

ObjectiveThis study was to identify small inhibitory RNAs (siRNAs) that are effective in inhibiting growth of cervical cancer cell lines harboring human papilloma virus (HPV) and to examine how siRNAs interact with interferon beta (IFN-β) and thimerosal.MethodsThe HPV18-positive HeLa and C-4I cell lines were used. Four types of siRNAs were designed according to their target (both E6 and E7 vs. E6 only) and sizes (21- vs. 27-nucleotides); Ex-18E6/21, Ex-18E6/27, Sp-18E6/21, and Sp-18E6/27. Each siRNA-transfected cells were cultured with or without IFN-b and thimerosal and their viability was measured.ResultsThe viabilities of HPV18-positive tumor cells were reduced by 21- and 27-nucleotide siRNAs in proportion to the siRNA concentrations. Of the two types of siRNAs, the 27-nucleotide siRNA constructs showed greater inhibitory efficacy. Sp-18E6 siRNAs, which selectively downregulates E6 protein only, were more effective than the E6- and E7-targeting Ex-18E6 siRNAs. siRNAs and IFN-β showed the synergistic effect to inhibit HeLa cell survival and the effect was proportional to both siRNA and IFN-β concentrations. Thimerosal in the presence of siRNA exerted a dose-dependent inhibition of C-4I cell survival. Finally, co-treatment with siRNA, IFN-β, and thimerosal induced the most profound decrease in the viability of both cell lines.ConclusionLong (27-nucleotides) siRNAs targeting E6-E7 mRNAs effectively reduce the viability of HPV18-positive cervical cancer cells and show the synergistic effect in combination with IFN-b and thimerosal. It is necessary to find the rational design of siRNAs and effective co-factors to eradicate particular cervical cancer.

Onset Time and Durability of Huntingtin Suppression in Rhesus Putamen After Direct Infusion of Antihuntingtin siRNA.

One possible treatment for Huntington's disease involves direct infusion of a small, interfering RNA (siRNA) designed to reduce huntingtin expression into brain tissue from a chronically implanted programmable pump. Here, we studied the suppression of huntingtin mRNA achievable with short infusion times, and investigated how long suppression may persist after infusion ceases. Rhesus monkeys received 3 days of infusion of Magnevist into the putamen to confirm catheter patency and fluid distribution. After a 1-week washout period, monkeys received radiolabeled siRNA targeting huntingtin. After 1 or 3 days of siRNA delivery, monkeys were either terminated, or their pumps were shut off and they were terminated 10 or 24 days later. Results indicate that the onset of huntingtin mRNA suppression in the rhesus putamen occurs rapidly, achieving a plateau throughout the putamen within 4 days. Conversely, loss of huntingtin suppression progresses slowly, persisting an estimated 27–39 days in the putamen and surrounding white matter. These findings indicate the rapid onset and durability of siRNA-mediated target gene suppression observed in other organs also occurs in the brain, and support the use of episodic delivery of siRNA into the brain for treatment of Huntington's disease and possibly other neurodegenerative diseases.

Nanolipoparticles-mediated MDR1 siRNA delivery: preparation, characterization and cellular uptake

Objective(s): Lipid-based nanoparticles (NLP) are PEGylated carriers composed of lipids and encapsulated nucleic acids with a diameter less than 100 nm. The presence of PEG in the NLP formulation improves the particle pharmacokinetic behavior. The purpose of this study was to prepare and characterize NLPs containing MDR1 siRNA and evaluate their cytotoxicity and cellular uptake. MDR1 siRNA could be used in multidrug resistance reversal in cancer therapy.Materials and Methods: siRNAs were encapsulated into NLPs consisted of mPEG-DSPE/DOTAP/DOPE (10:50:40 molar ratio) by the detergent dialysis method. The particle diameters of NLPs and their surface charge were measured using dynamic light scattering. siRNA encapsulation efficiency was determined by an indirect method via filtration and free siRNA concentration determination. NLPs cytotoxicity was investigated by MTT assay. The ability of NLPs for siRNA delivery checked in two human cell lines (MCF-7/ADR and EPP85-181/RDB) by fluorescence microscopy and compared with oligofectamine.Results: NLPs containing MDR1 siRNA were prepared with the stable size of 80-90 nm and the zeta potential near to neutral. The siRNA encapsulation efficacy was more than 80%. These properties are suitable for in vivo siRNA delivery. NLPs cytotoxicity studies demonstrated they were non-toxic at the doses used. NLPs improved siRNA localization in both cell lines.Conclusion: NLPs containing MDR1 siRNA can be a good candidate for in vivo siRNA delivery studies.

Induction function of siRNA-mediated survivin gene silencing on nasopharyngeal carcinoma cell apoptosis

We examined the function of survivin gene expression in patients with nasopharyngeal carcinoma (NPC), as well as small interfering RNA (siRNA) on controlling CNE-2 NPC proliferation and apoptosis. Immunohistological methods, in situ hybridization, and reverse transcription-polymerase chain reaction technique were used to detect survivin protein and mRNA expression. We designed an siRNA sequence to inhibit survivin gene expression. The MTT method was used to examine the function of siRNA on controlling cell growth and proliferation. Induction of cell apoptosis by siRNA was examined by flow cytometry; electron microscopy was used to observe ultrastructure changes in CNE-2 cells. Western blotting was used to detect survivin gene expression. The survivin protein was expressed in 71.9% of cells, while its mRNA was expressed in 65.6% of cells. Relative mRNA expression was 4.16 x 10(-2); these data for the control groups were 23.3, 33.3, and 4.42 x 10(-4), respectively. Following transfection with 3 different siRNA sequences, survivin mRNA expression in CNE-2 cells was decreased. Inhibition of cell proliferation and rate of apoptosis increased with increasing siRNA concentration. Western blotting revealed decreased survivin expression and electron microscopy revealed ultrastructural changes in cancer cells. Survivin gene expression in NPC generally increased. In vitro transcription of siRNA decreased CNE-2 survivin gene expression, and different sequences of siRNA decrease gene expression in CNE-2 cells to varying degrees. Transfected siRNA3 can effectively inhibit CNE-2 cell proliferation and induce apoptosis; gene silencing using siRNA may represent a new treatment for NPC.

A simple and efficient method to transfect small interference RNA into bovine SCNT embryos

RNA interference is an important tool to study the gene function. Microinjection and electroporation are usually used to transfer DNA, small interference RNA (siRNA), morpholinos, and protein into oocytes or embryos. This study used a simple and effective method to transfect siRNA into bovine somatic cell nuclear transfer (SCNT) embryos. In this method, siRNA transfection and electrofusion of SCNT were combined. A pair of platinum microelectrodes was used during SCNT to complete electrofusion. A CY3-labeled siRNA-targeted DNA methyltransferase-1 (DNMT1) was chosen to verify the siRNA transfection efficiency of this approach. First, a suitable concentration of siRNA was mixed with Zimmermann's fusion medium. Reconstructed embryos were then added into the microdrops of the mixed fusion medium to simultaneously transfect the siRNA and electrofuse the SCNT embryos. Our results showed that transfecting DNMT1 siRNA via the proposed method caused obvious CY3 fluorescence and significant downregulation of DNMT1 messenger RNA, DNMT1 protein, and global DNA methylation levels in the SCNT embryos. Meanwhile, the survival rate after electrofusion (90.4% vs. 89.4% vs. 89.1%, P > 0.05) and developmental rates of the SCNT embryos (72.8% vs. 74.9% vs. 72.4%, P > 0.05; 29.7% vs. 31.7% vs. 29.7%, P > 0.05) were not significantly affected. In summary, siRNAs were effectively transfected into the SCNT embryos via the proposed method and exert their functions, and the normal development of preimplantation SCNT embryos was not affected by the method used.

170 GENE SILENCING OF BMPRII IN BOVINE GRANULOSA CELLS

Granulosa cells (GC) are important constituents of the follicular environment for oocyte competence acquisition. However, their functionality depends on oocyte-derived factors, such as GDF9 and BMP15, which act through BMPRII receptor signalling. Gene silencing using lipofection has been used as an important tool to investigate the role of cell genes and proteins. The aim of this study was to establish the ideal conditions for lipofection in bovine GC and starting from this protocol to establish a methodology for silencing of the BMPRII gene by RNA interference, and to use this strategy to study the functions of BMPRII in GDF9 signalling. GC were obtained from slaughterhouse ovaries by aspiration of follicles (2 to 6 mm) and subsequently cultured in DMEM medium at 38.5°C and 5% CO2 in air. All data analyzes were performed by GraphPad Prism® version 5.0 software (GraphPad Software Inc., La Jolla, CA, USA), using one-way ANOVA followed by Tukey's test. For optimizing the conditions for lipofection, the GC were treated with different amounts of lipofection agents Lipofectamine® RNAiMAX (Invitrogen, Carlsbad, CA, USA; 1, 2, and 3 µL) or LipofectamineTM 2000 (Invitrogen; 1, 2, and 3 µL) and the transfection indicators Siglo® (GE Healthcare, Waukesha, WI, USA; 30 to 100 nM) or FUGW transgenic plasmid (100 to 900 nM) during 24 and 48 h. The highest efficiency of lipofection was observed at 24 h of culture with 2 μL of Lipofectamine™ 2000 + 100 nM of Siglo®. Based on these conditions, different concentrations of siBMPRII (100 to 500 pM) were tested during 24 h of culture and subsequently, different incubation times (0, 6, 12, 18, and 24 h) with the best siRNA concentration in order to establish optimum conditions for gene silencing. GC were evaluated for the relative abundance of mRNA for BMPRII using PPIA and β-actin as endogenous controls by real-time PCR. All concentrations provided similar and highly significant transcript reduction in comparison to control (P &lt; 0.001), so the lowest of them was adopted (100 pM). For different incubation times with 100 pM siBMPRII also a similar decrease was also seen, which was more significant at 24 h (P &lt; 0.01). The best concentration (100 pM) and incubation time (24 h) with the siRNA were analysed by Western blotting, which confirmed the BMPRII reduction also at protein level (P &lt; 0.05). For functional evaluation, GC submitted or no to gene silencing were incubated with or without GDF9 (100 ng) and assessed for expression of genes controlled by GDF9 through its BMPRII receptor (LHR, INHA, and INHBA), besides the CYP17 gene, a marker of potential theca cells contamination. LHR expression was reduced in silenced groups (P &lt; 0.05) and highly suppressed by GDF9 in non-silenced groups (P &lt; 0.001), while INHA and INHBA expression remained constant in the different groups (P &gt; 0.05), suggesting that the control of these genes in bovine does not behave as reported in other species. The lack of amplification CYP17 indicates the nonexistence of the contamination. In conclusion, this study allowed the establishment of an efficient methodology for the silencing of BMPRII by lipofection in bovine GC, which may be used as a tool for the functional study of BMPRII and potentially for other genes of interest. Research was supported by FAPESP and CNPq.

Inhibitory effect of bone marrow mesenchymal stem cells on lymphoma cell proliferation

Growing evidences show that mesenchymal stem cells (MSC) home to tumorgenesis and inhibit tumor cells, however, their molecular mechanisms are unclear. The purpose of this study was to explore the effect of B7-H4 in the influence of the mouse MSC on the proliferation of lymphoma cell line EL-4. The expression of B7-H4 on the MSC cell line C3H10T1/2 (C3H10) was detected by using immunofluorescence. FAM-siRNA was synthesized and transfected into C3H10 cells by INTERFER in (TM) siRNA Transfection Reagent. The transfection efficiency was determined by fluorescent microscopy and flow cytometry. The mRNA expression of B7-H4 was detected by RT-PCR after transfection of siRNA-B7-H4 into C3H10 cell line. The EL-4 was co-cultured with C3H10 siRNA-NC or C3H10 siRNA-B7-H4 for 48 hours, then was compared with EL-4 cultured alone, after 48 hours the cells were harvested under the confocal microscopy and measured by means of CCK-8 Kit. The results showed that the siRNA transfection efficiency in C3H10 cells reached to 72.43%, B7-H4 expressed highly on C3H10, the B7-H4 mRNA expression was down-regulated by transfection with different concentrations of siRNA into C3H10 cells. The proliferation of EL-4 was inhibited by C3H10 cells, and the effects were weakened and even disappeared after down-regulation of B7-H4. It is concluded that C3H10 can inhibit the proliferation of EL-4 through the expression of B7-H4, and this study provides new targets for the clinical treatment of lymphoma.

Light intensity and temperature affect systemic spread of silencing signal in transient agroinfiltration studies.

RNA silencing is a sequence-specific post-transcriptional gene inactivation mechanism that operates in diverse organisms and that can extend beyond its site of initiation, owing to the movement of the silencing signal, called non-autonomous gene silencing. Previous studies have shown that several factors manifest the movement of the silencing signal, such as the size (21 or 24 nucleotides) of the secondary small interfering RNA (siRNA) produced, the steady-state concentration of siRNAs and their cognate messenger RNA (mRNA) or a change in the sink-source status of plant parts affecting phloem translocation. Our study shows that both light intensity and temperature have a significant impact on the systemic movement of the silencing signal in transient agroinfiltration studies in Nicotiana benthamiana. At higher light intensities (≥ 450 μE/m(2)/s) and higher temperatures (≥ 30 °C), gene silencing was localized to leaf tissue that was infiltrated, without any systemic spread. Interestingly, in these light and temperature conditions (≥ 450 μE/m(2) /s and ≥ 30 °C), the N. benthamiana plants showed recovery from the viral symptoms. However, the reduced systemic silencing and reduced viral symptom severity at higher light intensities were caused by a change in the sink-source status of the plant, ultimately affecting the phloem translocation of small RNAs or the viral genome. In contrast, at lower light intensities (<300 μE/m(2)/s) with a constant temperature of 25 °C, there was strong systemic movement of the silencing signal in the N. benthamiana plants and reduced recovery from virus infections. The accumulation of gene-specific siRNAs was reduced at higher temperature as a result of a reduction in the accumulation of transcript on transient agroinfiltration of RNA interference (RNAi) constructs, mostly because of poor T-DNA transfer activity of Agrobacterium, possibly also accompanied by reduced phloem translocation.