Tool For Gene Silencing Research Articles

The siRNA Non-seed Region and Its Target Sequences Are Auxiliary Determinants of Off-Target Effects.

RNA interference (RNAi) is a powerful tool for post-transcriptional gene silencing. However, the siRNA guide strand may bind unintended off-target transcripts via partial sequence complementarity by a mechanism closely mirroring micro RNA (miRNA) silencing. To better understand these off-target effects, we investigated the correlation between sequence features within various subsections of siRNA guide strands, and its corresponding target sequences, with off-target activities. Our results confirm previous reports that strength of base-pairing in the siRNA seed region is the primary factor determining the efficiency of off-target silencing. However, the degree of downregulation of off-target transcripts with shared seed sequence is not necessarily similar, suggesting that there are additional auxiliary factors that influence the silencing potential. Here, we demonstrate that both the melting temperature (Tm) in a subsection of siRNA non-seed region, and the GC contents of its corresponding target sequences, are negatively correlated with the efficiency of off-target effect. Analysis of experimentally validated miRNA targets demonstrated a similar trend, indicating a putative conserved mechanistic feature of seed region-dependent targeting mechanism. These observations may prove useful as parameters for off-target prediction algorithms and improve siRNA ‘specificity’ design rules.

A Tandem Oligonucleotide Approach for SNP-Selective RNA Degradation Using Modified Antisense Oligonucleotides.

Antisense oligonucleotides have been studied for many years as a tool for gene silencing. One of the most difficult cases of selective RNA silencing involves the alleles of single nucleotide polymorphisms, in which the allele sequence is differentiated by a single nucleotide. A new approach to improve the performance of allele selectivity for antisense oligonucleotides is proposed. It is based on the simultaneous application of two oligonucleotides. One is complementary to the mutated form of the targeted RNA and is able to activate RNase H to cleave the RNA. The other oligonucleotide, which is complementary to the wild type allele of the targeted RNA, is able to inhibit RNase H cleavage. Five types of SNPs, C/G, G/C, G/A, A/G, and C/U, were analyzed within the sequence context of genes associated with neurodegenerative disorders such as Alzheimer’s disease, Parkinson’s disease, ALS (Amyotrophic Lateral Sclerosis), and Machado-Joseph disease. For most analyzed cases, the application of the tandem approach increased allele-selective RNA degradation 1.5–15 fold relative to the use of a single antisense oligonucleotide. The presented study proves that differentiation between single substitution is highly dependent on the nature of the SNP and surrounding nucleotides. These variables are crucial for determining the proper length of the inhibitor antisense oligonucleotide. In the tandem approach, the comparison of thermodynamic stability of the favorable duplexes WT RNA-inhibitor and Mut RNA-gapmer with the other possible duplexes allows for the evaluation of chances for the allele-selective degradation of RNA. A larger difference in thermodynamic stability between favorable duplexes and those that could possibly form, usually results in the better allele selectivity of RNA degradation.

Transformation of Ulva mutabilis (Chlorophyta) by vector plasmids integrating into the genome

A method for the stable transformation of the green marine macroalga Ulva mutabilis was developed based on vector plasmids integrating into the genome. By combination of the expression signals (promoter, enhancer, and transcriptional termination sequences) of a chromosomal rbcS gene from U.mutabilis with the bleomycin resistance gene (ble) from Streptoalloteichus hindustanus, a dominant selectable marker gene was constructed for the preparation of a series of E.coli-U.mutabilis shuttle vector plasmids. Special vectors were prepared for the introduction and expression of foreign genes in Ulva, for insertional mutagenesis and gene tagging by plasmid integration into the genome, and for protein tagging by the green fluorescent protein, as well as tools for posttranscriptional gene silencing and cosmid cloning to prepare genomic gene libraries for mutant gene complementation. The vectors were successfully tested in pilot experiments, where they were efficiently introduced into Ulva gametes, zoospores or protoplasts of somatic blade cells by treatment with Ca(2+) -ions and polyethylene glycol under isotonic conditions at low ionic strength. The parthenogenetically propagated phleomycin-resistant transformants of the mutant slender (sl) and the wildtype (wt) were demonstrated to be carrying the plasmids randomly integrated into the chromosomes often as tandem repeat clusters.

English

The cellular membrane constitutes an effective barrier that protects the complex, yet highly ordered, intracellular compartment of the cell. Passage of molecules across this barrier is highly regulated and highly restricted. Cell penetrating peptides (CPPs) are a class of small cationic peptides that are able to defy the rules of membrane passage and gain access to the intracellular environment. MPG is one member of this class of cell penetrating peptides. In this study, MPG was used to deliver double-stranded siRNA into human multipotent stem (hMADS) cells. Confluent and differentiated hMADS cells were transfected for 4 h with 30 nM siRNA-MPG-complex (SMC) and the controls were overlaid with free siRNA (SO) and analysed after 24, 48 and 72 h incubation post-transfection for internalization of the siRNA complex using Zeiss Axioimager. There was no noticeable adverse morphological variation between the siRNA-MPG complex transfected groups and the ‘free siRNA’ and the ‘cell only’ control groups. There was also no observed cytotoxicity associated with transfection. The differentiation of the cells into adipocyte phenotype was observed normally in all the groups. Bright fluorescence speckles were detected in the SMC transfected cells at all the 3 time points in the cytoplasm as well as in the nucleus 24 and 48 h post-transfection. The efficiency of delivery 24 h after transfection in the confluent cells was about 90%, in cells differentiating three days toward adipocytes about 80, and 50 to 60% of the cells showed internalised siRNA-MPG complex even 48 and 72 h post- transfection. This study shows that MPG efficiently mediated cytoplasmic and nuclear delivery of double-stranded siRNA into human mesenchymal stem cells under mild conditions in proliferating and in differentiation stages. These results demonstrate that MPG is a very effective and robust non-viral based transfection agent, easy to apply to non-dividing adherent cells in situ.  Thus, MPG is a valuable tool for transient gene and microRNA silencing in vitro. Key words: Cell-penetrating peptides, microRNA, MPG, siRNA, stem cells, transfection, delivery, nanoparticles.&nbsp

High Throughput Sequencing of Entamoeba 27nt Small RNA Population Reveals Role in Permanent Gene Silencing But No Effect on Regulating Gene Expression Changes during Stage Conversion, Oxidative, or Heat Shock Stress.

The human parasite Entamoeba histolytica has an active RNA interference (RNAi) pathway with an extensive repertoire of 27nt small RNAs that silence genes. However the role of this pathway in regulating amebic biology remains unknown. In this study, we address whether silencing via 27nt small RNAs may be a mechanism for controlling gene expression changes during conversion between the trophozoite and cyst stages of the parasite. We sequenced small RNA libraries generated from trophozoites, early cysts, mature cysts, and excysting cells and mapped them to the E. invadens genome. Our results show that, as in E. histolytica, small RNAs in E. invadens are largely ~27nt in length, have an unusual 5'-polyphosphate structure and mediate gene silencing. However, when comparing the libraries from each developmental time-point we found few changes in the composition of the small RNA populations. Furthermore, genes targeted by small RNAs were permanently silenced with no changes in transcript abundance during development. Thus, the E. invadens 27nt small RNA population does not mediate gene expression changes during development. In order to assess the generalizability of our observations, we examined whether small RNAs may be regulating gene expression changes during stress response in E. histolytica. Comparison of the 27nt small RNA populations from E. histolytica trophozoites from basal conditions, or after heat shock or exposure to oxidative stress showed few differences. Similar to data in E. invadens development, genes targeted by small RNAs were consistently silenced and did not change expression under tested stress conditions. Thus, the biological roles of the 27nt small RNA population in Entamoeba remain elusive. However, as the first characterization of the RNAi pathway in E. invadens these data serve as a useful resource for the study of Entamoeba development and open the door to the development of RNAi-based gene silencing tools in E. invadens.

Gold nanoparticle-siRNA mediated oncogene knockdown at RNA and protein level, with associated gene effects.

RNAi is a powerful tool for gene silencing that can be used to reduce undesirable overexpression of oncogenes as a novel form of cancer treatment. However, when using RNAi as a therapeutic tool there is potential for associated gene effects. This study aimed to utilize gold nanoparticles to deliver siRNA into HeLa cells. Knockdown of the c-myc oncogene by RNAi, at the RNA, protein and cell proliferation level was achieved, while also identifying associated gene responses. The gold nanoparticles used in this study present an excellent delivery platform for siRNA, but do note associated gene changes. The study highlights the need to more widely assess the cell physiological response to RNAi treatment, rather than focus on the immediate RNA levels.

Artificial MiRNA Knockdown of Platelet Glycoprotein lbα: A Tool for Platelet Gene Silencing.

In recent years, candidate genes and proteins implicated in platelet function have been identified by various genomic approaches. To elucidate their exact role, we aimed to develop a method to apply miRNA interference in platelet progenitor cells by using GPIbα as a proof-of-concept target protein. After in silico and in vitro screening of siRNAs targeting GPIbα (siGPIBAs), we developed artificial miRNAs (miGPIBAs), which were tested in CHO cells stably expressing GPIb-IX complex and megakaryoblastic DAMI cells. Introduction of siGPIBAs in CHO GPIb-IX cells resulted in 44 to 75% and up to 80% knockdown of GPIbα expression using single or combined siRNAs, respectively. Conversion of siGPIBAs to miGPIBAs resulted in reduced silencing efficiency, which could however be circumvented by tandem integration of two hairpins targeting different regions of GPIBA mRNA where 72% GPIbα knockdown was achieved. CHO GPIb-IX cells transfected with the miGPIBA construct displayed a significant decrease in their ability to aggregate characterized by lower aggregate numbers and size compared to control CHO GPIb-IX cells. More importantly, we successfully silenced GPIbα in differentiating megakaryoblastic DAMI cells that exhibited morphological changes associated with actin organization. In conclusion, we here report the successful use of miRNA technology to silence a platelet protein in megakaryoblastic cells and demonstrate its usefulness in functional assays. Hence, we believe that artificial miRNAs are suitable tools to unravel the role of a protein of interest in stem cells, megakaryocytes and platelets, thereby expanding their application to novel fields of basic and translational research.

Paired-termini antisense RNA mediated inhibition of DoxR in Streptomyces peucetius ATCC 27952

Our previous study provided an insight into DoxR as a negative regulator of doxorubicin production in Streptomyces peucetius ATCC 27952. Streptomyces hosts are advantageous in terms of producing a number of pharmaceuticals in low titer. Antisense RNAs (asRNAs) silencing strategy acts as an alternative tool for metabolic engineering of microorganisms for construction of an efficient cell factory. In this study, a paired-termini antisense RNAs (PTasRNAs) silencing strategy was employed for inhibition of DoxR to enhance doxorubicin production. To continue this endeavor, we designed and constructed the piBR702 vector for the expression of PTasRNAs in monocistronic mode. Further, two variants of asRNA, adoxR and bdoxR were designed and cloned into piBR702. All the rDNAs were transformed into S. peucetius to generate engineered strains. The engineered strains, S. peucetius A and S. peucetius B produced enhanced titers of doxorubicin, daunorubicin, and e-rhodomycinone; however, no such change was seen in S. peucetius AB. Moreover, RT-PCR analysis of doxR from S. peucetius A and S. peucetius B, together with the higher production from S. peucetius A, confirmed adoxR as a better asRNA than bdoxR. The reason behind this could be due to the simple secondary structure and low binding free energy of adoxR (−419 kcal/mol) than bdoxR (−358.8 kcal/mol). Our study demonstrated that antibiotic production was enhanced significantly by inhibiting DoxR, a negative regulator in S. peucetius using PTasRNAs. In addition, this study further provides an insight into PTasRNAs as an effective tool for gene silencing in Streptomyces and its use as an effective tool for metabolic engineering.

B-cell receptor-guided delivery of peptide-siRNA complex for B-cell lymphoma therapy.

BackgroundDespite the clinical response of conventional anticancer therapy, including chemotherapeutic treatments, radiation therapy and corticosteroids, tumorigenic B-cell lymphomas show an incomplete response to clinical practices that result in a minimal residual disease (MRD) where few residual neoplastic cells undetected in vivo, replenish the cancer cell reservoir. This scenario, which is also shared with other cancer diseases, requires the development of strategies to advance in novel, selective targeting toward the tumorigenic cells that survive to the anticancer agents.MethodsHere, we have taken advantage of the therapeutic properties of an idiotype specific peptide (pA20-36) that bind specifically to murine B-lymphoma cells in the setting of an anti cancer strategy, based on the selected delivery of electrostatic-based complex, peptide-siRNA. To this end, two engineered, arginine rich, peptides that included the pA20-36 targeting sequence were designed to bind fluorescent-labelled siRNA. One peptide presented 9 Arg at the C-terminal of pA20-36 whereas the other included 5 Arg at the N- and C-terminus, respectively.ResultsCompared to the control and random peptide-siRNA complexes, both pA20-36-siRNA complexes were endowed with the selective delivering of fluorescent-labelled siRNA toward the A20 murine B-cell lymphoma, as evaluated by cytofluorimetry and confocal microscopy, whereas fluorescent-labelled siRNA alone was not internalized in the selected cells. Compared to peptide controls, the use of the modified pA20-36 peptides complexed with siRNA anti-GAPDH and anti-Bcl2 showed a down-regulation in the expression levels of the corresponding genes.ConclusionsPeptide-siRNA complex can be suitable tool for both selective peptide-driven cell targeting and gene silencing. In this setting, the improvement of this strategy is expected to provide a safe and non-invasive approach for the delivery of therapeutic molecules.

Endocytic pathway mediates refractoriness of insect Bactrocera dorsalis to RNA interference.

RNA interference (RNAi) is a powerful and convenient tool for sequence-specific gene silencing, and it is triggered by double-stranded RNA (dsRNA). RNAi can be easily achieved in many eukaryotes by either injecting or feeding dsRNAs. This mechanism has demonstrated its potential in fundamental research on genetics, medicine and agriculture. However, the possibility that insects might develop refractoriness to RNAi remains unexplored. In this study, we report that the oriental fruit fly, Bactrocera dorsalis, became refractory to RNAi using orally administered dsRNA targeting endogenous genes. Furthermore, refractoriness to RNAi is not gene-specific, and its duration depends on the dsRNA concentration. RNAi blockage requires the endocytic pathway. Fluorescence microscopy indicated that in RNAi refractory flies, dsRNA uptake is blocked. Genes involved in the entry of dsRNAs into cells, including chc, cog3, light and others, are down-regulated in RNAi refractory flies. Increasing the endocytic capacity by improving F-actin polymerization disrupts RNAi refractoriness after both primary and secondary dsRNA exposures. Our results demonstrate that an insect can become refractory to RNAi by preventing the entry of dsRNA into its cells.

Improved design of PPRHs for gene silencing.

Nowadays, the modulation of gene expression by nucleic acids has become a routine tool in biomedical research for target validation and it is also used to develop new therapeutic approaches. Recently, we developed the so-called polypurine reverse Hoogsteen hairpins (PPRHs) that show high stability and a low immunogenic profile and we demonstrated their efficacy both in vitro and in vivo. In this work, we explored different characteristics of PPRHs to improve their usage as a tool for gene silencing. We studied the role of PPRH length in the range from 20 to 30 nucleotides. We also proved their higher affinity of binding and efficacy on cell viability compared to nonmodified TFOs. To overcome possible off-target effects, we tested wild-type PPRHs, which proved to be capable of binding to their target sequence with more affinity, displaying a higher stability of binding and a higher effect in terms of cell viability. Moreover, we developed a brand new molecule called Wedge-PPRH with the ability to lock the ds-DNA into the displaced structure and proved its efficacy in prostate and breast cancer cell lines.

Epigenetic transitions leading to heritable, RNA-mediated de novo silencing in Arabidopsis thaliana

In plants, RNA-directed DNA methylation (RdDM), a mechanism where epigenetic modifiers are guided to target loci by small RNAs, plays a major role in silencing of transposable elements (TEs) to maintain genome integrity. So far, two RdDM pathways have been identified: RNA Polymerase IV (PolIV)-RdDM and RNA-dependent RNA Polymerase 6 (RDR6)-RdDM. PolIV-RdDM involves a self-reinforcing feedback mechanism that maintains TE silencing, but cannot explain how epigenetic silencing is first initiated. A function of RDR6-RdDM is to reestablish epigenetic silencing of active TEs, but it is unknown if this pathway can induce DNA methylation at naïve, non-TE loci. To investigate de novo establishment of RdDM, we have used virus-induced gene silencing (VIGS) of an active flowering Wageningen epiallele. Using genetic mutants we show that unlike PolIV-RdDM, but like RDR6-RdDM, establishment of VIGS-mediated RdDM requires PolV and DRM2 but not Dicer like-3 and other PolIV pathway components. DNA methylation in VIGS is likely initiated by a process guided by virus-derived small (s) RNAs that are 21/22-nt in length and reinforced or maintained by 24-nt sRNAs. We demonstrate that VIGS-RdDM as a tool for gene silencing can be enhanced by use of mutant plants with increased production of 24-nt sRNAs to reinforce the level of RdDM.

Abstract 461: Modulation of estrogen receptor (ER) and androgen receptor (AR) by a modified CRISPR-Cas9 system

Abstract Clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated (Cas) system is a bacterial adaptive immune defense against foreign nucleic acids from viruses, plasmids, and other mobile genetic elements. There are three types of CRISPR systems; type II uses single effector enzyme, Cas9, to cleave double-stranded DNA (dsDNA). Because of this unique feature, CRISPR/Cas9 system has been widely used as a genetic engineering tool from bacteria to mammals. In this system, Cas9 nuclease can bind and cleave genome directed by sequence-specific guide RNA (gRNA). While the wild type Cas9 can cleave double stranded DNA, the mutant Cas9 at D10A and H804A can only bind to but cannot cleave the DNA. Given this unique feature, we tried to repurpose Cas9 as a gene activation or suppression tool. To achieve this, we generated DMCas9 (double-mutant Cas9), and fused it with VP16AD (VP16 activation domain) or VP64AD (VP64 activation domain) to explore its utility for gene activation. To generate a Cas9-based gene silencing tool, we fused DMCas9 with Krab, a well-known protein domain with suppression function. Using a reporter system carrying a specific sequence that was recognized by a given gRNA and mCherry as a readout, we demonstrated that co-expression of VP16AD-DMCas9 and specific gRNAs leads to a at least three-fold induction of mCherry signal; in contrast, DMhCas9-Krab along with the specific gRNA resulted in a significant suppression of mCherry activity. Furthermore, we showed that DMCas9-Krab combined with gRNAs suppressed endogenous expression of AR and ER in MCF-7 cells, causing cell growth inhibition. Together, these results demonstrate the feasibility of Cas9-based gene activation and gene suppression. Citation Format: Nanjiang Zhou, Yin-Yuan Mo. Modulation of estrogen receptor (ER) and androgen receptor (AR) by a modified CRISPR-Cas9 system. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 461. doi:10.1158/1538-7445.AM2014-461

SiRNA-based therapies for pulmonary diseases.

Various delivery strategies, involving siRNA as a therapeutic tool for gene silencing, have been highlighted through several investigations all over the world. One such medical target, where the siRNA-based therapies have been immensely explored and have met with considerable success, is the area of pulmonary disorders. Lung diseases have presented themselves as attractive targets for studying siRNA-mediated cures due to their widespread persistence and lethality. Another interesting feature in this case is that the lung is accessible to therapeutic agents via multiple administration routes including the nasal, oral and intravenous routes. Recent advances in pulmonary delivery highlight the exploitation of all these routes for administration of siRNA-based therapies, particularly by employing non-viral carriers like nanoparticles. Through this manuscript we aim to provide a comprehensive overview of the most important and the latest developments in non-viral siRNA delivery for lung diseases. We have focused our discussion on the diverse systems, which have been investigated for a plethora of pulmonary disorders, ranging from inflammatory conditions like asthma and COPD, to infectious diseases like tuberculosis, and to lung cancer. An overview of the preclinical and clinical investigations conducted in this area has also been presented to the readers. While a variety of these systems have been found to be promising in pre-clinical studies, their successful transition as therapeutics will ultimately depend upon their clinical safety and efficacy, as well as the specificity of the carriers and methods employed for their administration.

Lessons learned from vivo-morpholinos: How to avoid vivo-morpholino toxicity.

Vivo-morpholinos are a promising tool for gene silencing. These oligonucleotide analogs transiently silence genes by blocking either translation or pre-mRNA splicing. Little to no toxicity has been reported for vivo-morpholino treatment. However, in a recent study conducted in our lab, treatment of mice with vivo-morpholinos resulted in high mortality rates. We hypothesized that the deaths were the result of oligonucleotide hybridization, causing an increased cationic charge associated with the dendrimer delivery moiety of the vivo-morpholino. The cationic charge increased blood clot formation in whole blood treated with vivo-morpholinos, suggesting that clotting could have caused cardiac arrest in the deceased mice. Therefore, we investigate the mechanism by which some vivo-morpholinos increase mortality rates and propose techniques to alleviate vivo-morpholino toxicity.

Use of Peptide Nucleic Acids to Manipulate Gene Expression in the Malaria Parasite Plasmodium falciparum

One of the major concerns in treating malaria by conventional small drug molecules is the rapid emergence of drug resistance. Specific silencing of essential genes by antisense oliogomers has been proposed as an alternative approach that may result in antimalarial activity which is not associated with drug resistance. In addition, such an approach could be an important biological tool for studying many genes' function by reverse genetics. Here we present a novel methodology of using peptide nucleic acids (PNAs) as a useful tool for gene silencing in Plasmodium falciparum. PNAs, designed as specific antisense molecules, were conjugated to a cell penetrating peptide (CPP); namely, octa-D-lysine via the C-terminus, to allow facile delivery through cell membranes. PNAs added to P. falciparum cultures were found exclusively in infected erythrocytes and were eventually localized in nuclei of the parasites at all stages of intra erythrocytic development. We show that these PNAs specifically down regulated both a stably expressed transgene as well as an endogenous essential gene, which significantly reduced parasites' viability. This study paves the way for a simple approach to silence a variety of P. falciparum genes as means of deciphering their function and potentially to develop highly specific and potent antimalarial agents.

Functional nanostructures for effective delivery of small interfering RNA therapeutics.

Small interfering RNA (siRNA) has proved to be a powerful tool for target-specific gene silencing via RNA interference (RNAi). Its ability to control targeted gene expression gives new hope to gene therapy as a treatment for cancers and genetic diseases. However, siRNA shows poor pharmacological properties, such as low serum stability, off-targeting, and innate immune responses, which present a significant challenge for clinical applications. In addition, siRNA cannot cross the cell membrane for RNAi activity because of its anionic property and stiff structure. Therefore, the development of a safe, stable, and efficient system for the delivery of siRNA therapeutics into the cytoplasm of targeted cells is crucial. Several nanoparticle platforms for siRNA delivery have been developed to overcome the major hurdles facing the therapeutic uses of siRNA. This review covers a broad spectrum of non-viral siRNA delivery systems developed for enhanced cellular uptake and targeted gene silencing in vitro and in vivo and discusses their characteristics and opportunities for clinical applications of therapeutic siRNA.

Cardiovascular RNA Interference Therapy

Understanding of the roles of noncoding RNAs (ncRNAs) within complex organisms has fundamentally changed. It is increasingly possible to use ncRNAs as diagnostic and therapeutic tools in medicine. Regarding disease pathogenesis, it has become evident that confinement to the analysis of protein-coding regions of the human genome is insufficient because ncRNA variants have been associated with important human diseases. Thus, inclusion of noncoding genomic elements in pathogenetic studies and their consideration as therapeutic targets is warranted. We consider aspects of the evolutionary and discovery history of ncRNAs, as far as they are relevant for the identification and selection of ncRNAs with likely therapeutic potential. Novel therapeutic strategies are based on ncRNAs, and we discuss here RNA interference as a highly versatile tool for gene silencing. RNA interference-mediating RNAs are small, but only parts of a far larger spectrum encompassing ncRNAs up to many kilobasepairs in size. We discuss therapeutic options in cardiovascular medicine offered by ncRNAs and key issues to be solved before clinical translation. Convergence of multiple technical advances is highlighted as a prerequisite for the translational progress achieved in recent years. Regarding safety, we review properties of RNA therapeutics, which may immunologically distinguish them from their endogenous counterparts, all of which underwent sophisticated evolutionary adaptation to specific biological contexts. Although our understanding of the noncoding human genome is only fragmentary to date, it is already feasible to develop RNA interference against a rapidly broadening spectrum of therapeutic targets and to translate this to the clinical setting under certain restrictions.

RNAi gene therapy of SiHa cells via targeting human TERT induces growth inhibition and enhances radiosensitivity

Telomerase activity (TA) is reactivated in more than 90% of all human malignant tumors and normal somatic cells that lack TA. Thus, human TERT (hTERT) is a promising target in malignant tumor treatment. RNA interference is a powerful tool for gene silencing. In this study, we constructed siRNA#1-4 to knock down hTERT. All siRNAs were able to downregulate hTERT differently and we chose siRNA#3 (most effectively) in the following experiments. We studied the effects on cell proliferation, cell cycle, cell apoptosis and radiosensitivity using SiHa cells. Our results showed that siRNA#3 was able to silence hTERT gene effectively. The silencing of hTERT could induce immediate growth arrest, enhance the S phase in cell cycle study and lead to early apoptosis in human cervical cancer cells (SiHa). In clonogenic assays, we used multitarget-single hit and linear-quadratic models to assess the radiosensitivity after knockdown of hTERT. All results of parameters (D0, Dq, α, β) indicated that downregulation of hTERT enhanced radiosensitivity in SiHa cells.

Targeting cancer cell-specific RNA interference by siRNA delivery using a complex carrier of affibody-displaying bio-nanocapsules and liposomes

BackgroundSmall interfering RNA (siRNA) has attracted attention in the field of nucleic acid medicine as a RNA interference (RNAi) application that leads to gene silencing due to specific messenger RNA (mRNA) destruction. However, since siRNA is unstable in blood and unable to cross the cell membrane, encapsulation of siRNA into a carrier is required.ResultsIn this study, we used a carrier that combined ZHER2-displaying bio-nanocapsule (derived from hepatitis B virus surface antigen) and liposomes in a complex in order to investigate the feasibility of effective and target-cell-specific RNAi applications. As a result, by observing RNAi only in HER2-expressing breast cancer cells, using our proposed methodology, we successfully demonstrated target-cell-specific delivery and effective function expression of siRNA.ConclusionsThese findings show that, in the field of nucleic acid medicine, ZHER2-BNC/LP can be a useful carrier for siRNA delivery, and could also become a useful tool for gene silencing and to accomplish protein knock-down.