Effective siRNA Research Articles

Fluorescence Lifetime Imaging Microscopy (FLIM) of Intracellular Transport by Means of Doubly Labelled siRNA Architectures.

For monitoring the intracellular pathway of small interfering RNA (siRNA), both strands were labelled at internal positions by two ATTO dyes as an interstrand Förster resonance energy transfer pair. siRNA double strands show red emission and a short donor lifetime as readout, whereas siRNA antisense single strands show green emission and a long donor lifetime. This readout signals if GFP silencing can be expected (green) or not (red). We attached both dyes to three structurally different alkyne anchors by postsynthetic modifications. There is only a slight preference for the ribofuranoside anchors with the dyes at their 2’‐positions. For the first time, the delivery and fate of siRNA in live HeLa cells was tracked by fluorescence lifetime imaging microscopy (FLIM), which revealed a clear relationship between intracellular transport using different transfection methods and knockdown of GFP expression, which demonstrates the potential of our siRNA architectures as a tool for future development of effective siRNA.

Guanidinylated Cyclic Synthetic Polypeptides Can Effectively Deliver siRNA by Mimicking the Biofunctions of Both Cell-Penetrating Peptides and Nuclear Localization Signal Peptides.

Preventing endosomal entrapment of gene/vector nanocomplexes (NCs) remains a challenge for highly effective siRNA delivery. To address this problem, guanidinylated cyclic synthetic polypeptides (GCSPs) were synthesized using an efficient and easy method. GCSPs can condense siRNAs into NCs with an encapsulation efficiency of approximately 90%, over twice the effectiveness of Lipofectamine2000 (Lipo2000). The NCs can also mediate luciferase knockdown in HeLa cells with a silencing efficiency of 80%, nearly 2- and 1.1-fold that of Lipo2000 and PEI, respectively. More importantly, the NCs can enter cells by mimicking the bioactivity of cell-penetrating peptides (CPPs). NCs can also exert a nuclear localized function similar to nuclear localization signal peptides (NLSPs). Both biofunctions are helpful for preventing the common endosomal entrapment of NCs and greatly enhance the efficiency of siRNA delivery.

Designing an effective therapeutic siRNA to silence RdRp gene of SARS-CoV-2.

The devastating outbreak of COVID-19 has spread all over the world and has become a global health concern. There is no specific therapeutics to encounter the COVID-19. Small interfering RNA (siRNA)-based therapy is an efficient strategy to control human viral infections employing post-transcriptional gene silencing (PTGS) through neutralizing target complementary mRNA. RNA-dependent RNA polymerase (RdRp) encoded by the viral RdRp gene as a part of the replication-transcription complex can be adopted as an acceptable target for controlling SARS-CoV-2 mediated infection. Therefore, in the current study, accessible siRNA designing tools, including significant algorithms and parameters, were rationally used to design the candidate siRNAs against SARS-COV-2 encoded RdRp. The designed siRNA molecules possessed adequate nucleotide-based and other features for potent gene silencing. The targets of the designed siRNAs revealed no significant matches within the whole human genome, ruling out any possibilities for off-target silencing by the siRNAs. Characterization with different potential parameters of efficacy allowed selecting the finest siRNA among all the designed siRNA molecules. Further, validation assessment and target site accessibility prediction also rationalized the suitability of this siRNA molecule. Molecular docking study between the selected siRNA molecule and component of RNA interference (RNAi) pathway gave an excellent outcome. Molecular dynamics of two complexes: siRNA and argonaute complex, guide RNA, and target protein complex, have shown structural stability of these proteins. Therefore, the designed siRNA molecule might act as an effective therapeutic agent against the SARS-CoV-2 at the genome level and can prevent further outbreaks of COVID-19 in humans.

Optimization of PEGylated KL4 Peptide for siRNA Delivery with Improved Pulmonary Tolerance.

Pulmonary delivery of small interfering RNA (siRNA) is a promising therapeutic strategy for treating various respiratory diseases but an effective carrier for the delivery of siRNA into the cells of the lungs and a robust gene-silencing effect is still lacking. Previously, we reported that the KL4 peptide, a synthetic cationic peptide with a repeating KLLLL sequence, can mediate effective siRNA transfection in lung epithelial cells but its high hydrophobic leucine content, and hence poor water solubility, limits its application as a delivery vector. Here, we show that the covalent attachment of monodisperse poly(ethylene glycol) (PEG) improves the solubility of KL4 and the uptake of its complex with siRNA into lung epithelial cells, such that very robust silencing is produced. All PEGylated KL4 peptides, with PEG length varying between 6 and 24 monomers, could bind and form nanosized complexes with siRNA, but the interaction between siRNA and peptides became weaker as the PEG chain length increased. All PEGylated KL4 peptides exhibited satisfactory siRNA transfection efficiency on three human lung epithelial cell lines, including A549 cells, Calu-3 cells, and BEAS-2B cells. The PEG12KL4 peptide, which contains 12 monomers of PEG, was optimal for siRNA delivery and also demonstrated a low risk of inflammatory response and toxicity in vivo following pulmonary administration.

Extension in the approaches to treat cancer through siRNA system: a beacon of hope in cancer therapy

Along with the evolutionary breakthrough of RNA interference and the applicability for gene knockdown, a subsequent development in siRNA-based therapeutics has been attained. The gene therapy based on RNAi is in transition progress from the research aspects to clinical base. Being a potent tool, siRNA is used as therapeutic against several disorders. Cancer which is one of the deadliest diseases is now treated with an advanced mechanism of siRNA delivery inside the genome, leading to gene silencing; thereby, blocking translation of gene to form protein. siRNA tool delivers remedial effects with the advantages of safe delivery and efficiency. Despite its merits, barriers including instability at physiological conditions, lack of ability to cross biological membranes, off-targets, and safety are also associated with siRNA delivery system. The gene silencing efficiency values both in vitro and in vivo reported in the past years have been reviewed by material type (lipid, polymer, silica, porous silicon, and metal). This review presents a deep insight in the development of targeted delivery of siRNA. Since several clinical trials have also been performed regarding the siRNA delivery against cancer, it can also be stated that the delivery system should be good enough to achieve effective siRNA drug development.

Single-cell analysis reveals effective siRNA delivery in brain tumors with microbubble-enhanced ultrasound and cationic nanoparticles.

RNA-based therapies offer unique advantages for treating brain tumors. However, tumor penetrance and uptake are hampered by RNA therapeutic size, charge, and need to be "packaged" in large carriers to improve bioavailability. Here, we have examined delivery of siRNA, packaged in 50-nm cationic lipid-polymer hybrid nanoparticles (LPHs:siRNA), combined with microbubble-enhanced focused ultrasound (MB-FUS) in pediatric and adult preclinical brain tumor models. Using single-cell image analysis, we show that MB-FUS in combination with LPHs:siRNA leads to more than 10-fold improvement in siRNA delivery into brain tumor microenvironments of the two models. MB-FUS delivery of Smoothened (SMO) targeting siRNAs reduces SMO protein production and markedly increases tumor cell death in the SMO-activated medulloblastoma model. Moreover, our analysis reveals that MB-FUS and nanoparticle properties can be optimized to maximize delivery in the brain tumor microenvironment, thereby serving as a platform for developing next-generation tunable delivery systems for RNA-based therapy in brain tumors.

SNAP25 protects primary cortical neurons from hypoxic-ischemic injury associated with CREB signal.

Hypoxic-ischemic encephalopathy (HIE) could induce exacerbated changes and unpredictable effects in brain cells, and the mechanism remains unclear. HIE model was established in neonatal rats, Zea-Longa score and TTC staining were used to observe the neurobehavior and brain infarct volume in rats subjected to cerebral hypoxia-ischemia (HI). Primary cortical neurons were then cultured in vitro to establish an oxygen and glucose deprivation model. To determine the role of synaptosomal-associated protein-25 (SNAP25) in HIE, PC12 cells were cultured and effective siRNA fragments were screened, and SNAP25 was transfected into primary neurons. Then, quantitative real-time polymerase chain reaction was used to detect the mRNA expression level and immunofluorescence staining was used to observe the morphological changes of neurons before and after the injury. Finally, the abundance values of SNAP25 and its associated genes were filtered using the NCBI and GeneMANIA, respectively. HI leads to a decrease in neuronal number and an increase in SNAP25 expression. Whereas, the interference of SNAP25 caused marked decrease in neuronal number and the length of neurite. Moreover, the expression levels of CREB and SYP were significantly decreased after interference of SNAP25. SNAP25 exhibited several neuroprotective effects to neuronal protection in neonatal cerebral HI by regulating CREB and SYP.

Regulating the immunosuppressive tumor microenvironment to enhance breast cancer immunotherapy using pH-responsive hybrid membrane-coated nanoparticles

The combination of an immuno-metabolic adjuvant and immune checkpoint inhibitors holds great promise for effective suppression of tumor growth and invasion. In this study, a pH-responsive co-delivery platform was developed for metformin (Met), a known immuno-metabolic modulator, and short interfering RNA (siRNA) targeting fibrinogen-like protein 1 mRNA (siFGL1), using a hybrid biomimetic membrane (from macrophages and cancer cells)-camouflaged poly (lactic-co-glycolic acid) nanoparticles. To improve the endo-lysosomal escape of siRNA for effective cytosolic siRNA delivery, a pH-triggered CO2 gas-generating nanoplatform was developed using the guanidine group of Met. It can react reversibly with CO2 to form Met-CO2 for the pH-dependent capture/release of CO2. The introduction of Met, a conventional anti-diabetic drug, promotes programmed death-ligand 1 (PD-L1) degradation by activating adenosine monophosphate-activated protein kinase, subsequently blocking the inhibitory signals of PD-L1. As a result, siFGL1 delivery by the camouflaged nanoparticles of the hybrid biomimetic membrane can effectively silence the FGL1 gene, promoting T-cell-mediated immune responses and enhancing antitumor immunity. We found that a combination of PD-L1/programmed death 1 signaling blockade and FGL1 gene silencing exhibited high synergistic therapeutic efficacy against breast cancer in vitro and in vivo. Additionally, Met alleviated tumor hypoxia by reducing oxygen consumption and inducing M1-type differentiation of tumor-related macrophages, which improved the tumor immunosuppressive microenvironment. Our results indicate the potential of hybrid biomimetic membrane-camouflaged nanoparticles and combined Met-FGL1 blockade in breast cancer immunotherapy.

Positive-charge tuned gelatin hydrogel-siSPARC injectable for siRNA anti-scarring therapy in post glaucoma filtration surgery

Small interfering RNA (siRNA) therapy is a promising epigenetic silencing strategy. However, its widespread adoption has been severely impeded by its ineffective delivery into the cellular environment. Here, a biocompatible injectable gelatin-based hydrogel with positive-charge tuned surface charge is presented as an effective platform for siRNA protection and delivery. We demonstrate a two-step synthesis of a gelatin-tyramine (Gtn-Tyr) hydrogel with simultaneous charge tunability and crosslinking ability. We discuss how different physiochemical properties of the hydrogel interact with siSPARC (siRNA for secreted protein, acidic and rich in cysteine), and study the positive-charge tuned gelatin hydrogel as an effective delivery platform for siSPARC in anti-fibrotic treatment. Through in vitro studies using mouse tenon fibroblasts, the positive-charge tuned Gtn-Tyr hydrogel shows sustained siSPARC cellular internalization and effective SPARC silencing with excellent biocompatibility. Similarly, the same hydrogel platform delivering siSPARC in an in vivo assessment employing a rabbit model shows an effective reduction in subconjunctival scarring in post glaucoma filtration surgery, and is non-cytotoxic compared to a commonly used anti-scarring agent, mitomycin-C. Overall, the current siRNA delivery strategy involving the positive-charge tuned gelatin hydrogel shows effective delivery of gene silencing siSPARC for anti-fibrotic treatment. The current charge tunable hydrogel delivery system is simple to fabricate and highly scalable. We believe this delivery platform has strong translational potential for effective siRNA delivery and epigenetic silencing therapy.

Development of a Liquid Crystal Formulation that Can Penetrate the Stratum Corneum for Intradermal Delivery of Small Interfering RNA.

Topical delivery of small interfering RNA (siRNA) can be an attractive method for the treatment of skin diseases and improving the quality of life of patients. However, it is difficult for siRNA to pass through the two major barriers of the skin: the stratum corneum (SC) and tight junctions. We have previously reported that atopic dermatitis of skin without the SC can be efficiently treated by the intradermal administration of trans-activator of transcription (Tat) peptide and AT1002 (tight junction opening peptide). However, novel drug delivery systems are needed for effective SC penetration. Therefore, in the present study, we aimed to develop a lyotropic liquid crystalline (LC) system containing Tat and AT1002 for effective siRNA penetration through the SC. An LC formulation was prepared using selachyl alcohol and purified water, and its skin penetration ability was evaluated. No fluorescence was observed in mouse skin treated with a siRNA solution, as there was no intradermal localization of siRNA from naked siRNA. However, intradermal delivery of siRNA was remarkable and extensive with the LC formulation containing both Tat and AT1002. Semiquantitative analysis by brightness measurement revealed that the LC formulation containing both Tat and AT1002 had significantly enhanced intact skin permeability than other formulations. These results show that the functional peptides in the LC formulation increased SC penetration and intradermal delivery in the healthy skin. Therefore, this novel LC system may be useful in the treatment of various skin diseases.

Delivery of siRNA to the Eye: Protocol for a Feasibility Study to Assess Novel Delivery System for Topical Delivery of siRNA Therapeutics to the Ocular Surface.

Drug delivery to the eye remains a real challenge due to the presence of ocular anatomical barriers and physiological protective mechanisms. The lack of effective siRNA delivery mechanism has hampered the real potential of RNAi therapy, but recent literature suggests that nanocarrier systems show great promise in enhancing siRNA bioavailability and reducing the need for repeated intraocular injections. A diverse range of materials are under exploration worldwide, including natural and synthetic polymers, liposomes, peptides, and dendrimeric nanomaterials. This chapter describes a simple workflow for feasibility assessment of a proposed ocular surface siRNA delivery system. Gel retardation assay is used for investigation of optimal siRNA to carrier loading ratio. Fluorescent siRNA allows for initial in vitro testing of cellular uptake to corneal epithelial cells and investigation of in vivo siRNA delivery into mouse cornea by live animal imaging and fluorescence microscopy.

A functional and self-assembling octyl-phosphonium-tagged esculetin as an effective siRNA delivery agent.

Herein, we document a self-assembling octyl-TPP tagged esculetin (Mito-Esc) as functionally active and as a novel small molecule siRNA delivery vector. While Mito-Esc itself induces selective breast cancer cell death, the amphiphilic nature of Mito-Esc delivers therapeutic siRNAs intracellularly without the need for any excipient to exacerbate the anti-proliferative effects.

Hyperbranched Cationic Glycogen Derivative-Mediated IκBα Gene Silencing Regulates the Uveoscleral Outflow Pathway in Rats.

The role of the IκB/NF-κB signaling pathway in the uveoscleral outflow pathway was investigated with IκBα gene silencing mediated by the 3-(dimethylamino)-1-propylamine-conjugated glycogen (DMAPA-Glyp) derivative. The IκBα-siRNA-loaded DMAPA-Glyp complex was transfected into the ciliary muscles of rats by intracameral injection (labeled as the DMAPA-Glyp+siRNA group). The Lipofectamine™ 2000 (Lipo)/siRNA complex and the naked siRNA were set as the controls. The mRNA and protein expression of IκBα, NF-κBp65, and MMP-2 were analyzed by real-time PCR, western blotting, and in situ gelatin zymography. Nuclear translocation of NF-κBp65 was analyzed by immunofluorescence. Rat intraocular pressure (IOP) was monitored pre- and postinjection. Gene transfection efficiency and toxicity of the DMAPA-Glyp derivative were also evaluated. After RNA interference (RNAi), IκBα mRNA and protein expression were significantly inhibited. NF-κBp65 mRNA and protein expression showed no significant differences. Nevertheless, nuclear translocation of NF-κBp65 occurred in the DMAPA-Glyp+siRNA group. Both mRNA expression and activity of MMP-2 increased, with the largest increase in the DMAPA-Glyp+siRNA group. IOP in the DMAPA-Glyp+siRNA group fell to the lowest level on day 3 after RNAi. The levels of Cy3-siRNA in the ciliary muscle of the DMAPA-Glyp+siRNA group did not significantly decrease over time. At 7 and 14 d after RNAi, no significant pathological damage was detectable in the eyes injected with the DMAPA-Glyp derivative or the DMAPA-Glyp/siRNA complex. Taken together, our results suggest that downregulation of IκBα expression in the ciliary muscle plays a crucial role in reducing the IOP values of rats. IκBα may become a new molecular target for lowering IOP in glaucoma. The DMAPA-Glyp derivative is safe and feasible as an effective siRNA vector in rat eyes.

Synthesis and use of an amphiphilic dendrimer for siRNA delivery into primary immune cells.

Using siRNAs to genetically manipulate immune cells is important to both basic immunological studies and therapeutic applications. However, siRNA delivery is challenging because primary immune cells are often sensitive to the delivery materials and generate immune responses. We have recently developed an amphiphilic dendrimer that is able to deliver siRNA to a variety of cells, including primary immune cells. We provide here a protocol for the synthesis of this dendrimer, as well as siRNA delivery to immune cells such as primary T and B cells, natural killer cells, macrophages, and primary microglia. The dendrimer synthesis entails straightforward click coupling followed by an amidation reaction, and the siRNA delivery protocol requires simple mixing of the siRNA and dendrimer in buffer, with subsequent application to the primary immune cells to achieve effective and functional siRNA delivery. This dendrimer-mediated siRNA delivery largely outperforms the standard electroporation technique, opening a new avenue for functional and therapeutic studies of the immune system. The whole protocol encompasses the dendrimer synthesis, which takes 10 days; the primary immune cell preparation, which takes 3-10 d, depending on the tissue source and cell type; the dendrimer-mediated siRNA delivery; and subsequent functional assays, which take an additional 3-6 d.

An ionizable supramolecular dendrimer nanosystem for effective siRNA delivery with a favorable safety profile

Gene therapy using small interfering RNA (siRNA) is emerging as a novel therapeutic approach to treat various diseases. However, safe and efficient siRNA delivery still constitutes the major obstacle for clinical implementation of siRNA therapeutics. Here we report an ionizable supramolecular dendrimer vector, formed via self-assembly of a small amphiphilic dendrimer, as an effective siRNA delivery system with a favorable safety profile. By virtue of the ionizable tertiary amine terminals, the supramolecular dendrimer has a low positively charged surface potential and no notable cytotoxicity at physiological pH. Nonetheless, this ionizable feature imparted sufficient surface charge to the supramolecular dendrimer to enable formation of a stable complex with siRNA via electrostatic interactions. The resulting siRNA/dendrimer delivery system had a surface charge that was neither neutral, thus avoiding aggregation, nor too high, thus avoiding cytotoxicity, but was sufficient for favorable cellular uptake and endosomal release of the siRNA. When tested in different cancer cell lines and patient-derived cancer organoids, this dendrimer-mediated siRNA delivery system effectively silenced the oncogenes Myc and Akt2 with a potent antiproliferative effect, outperforming the gold standard vector, Lipofectamine 2000. Therefore, this ionizable supramolecular dendrimer represents a promising vector for siRNA delivery. The concept of supramolecular dendrimer nanovectors via self-assembly is new, yet easy to implement in practice, offering a new perspective for supramolecular chemistry in biomedical applications.

Amphiphilic peptide dendrimer-based nanovehicles for safe and effective siRNA delivery

Small interfering RNA (siRNA)-based RNA interference has emerged as a promising therapeutic strategy for the treatment of a wide range of incurable diseases. However, the safe and effective delivery of siRNA therapeutics into the interior of target cells remains challenging. Here, we disclosed novel amphiphilic peptide dendrimers (AmPDs) that composed of hydrophobic two lipid-like alkyl chains and hydrophilic poly(lysine) dendrons with different generations (2C18-KK2 and 2C18-KK2K4) as nanovehicles for siRNA delivery. These AmPDs are able to self-assemble into supramolecular nanoassemblies that are capable of entrapping siRNA molecules into nanoparticles to protect siRNA from enzymatic degradation and promote efficient intracellular uptake without evident toxicity. Interestingly, by virtue of the optimal balance of hydrophobic lipid-like entity and hydrophilic poly(lysine) dendron generations, AmPD 2C18-KK2K4 bearing bigger hydrophilic dendron can package siRNA to form stable, but more ready to disassemble complexes, thereby resulting in more efficient siRNA releasing and better gene silencing effect in comparison with AmPD 2C18-KK2 bearing smaller dendron. Additional studies confirmed that 2C18-KK2K4 can capitalize on the advantages of lipid and peptide dendrimer vectors for effective siRNA delivery. Collectively, our AmPD-based nanocarriers indeed represent a safe and effective siRNA delivery system. Our findings also provide a new perspective on the modulation of self-assembly amphiphilic peptide dendrimers for the functional and adaptive delivery of siRNA therapeutics.

Knockdown of ASH1L methyltransferase induced apoptosis inhibiting proliferation and H3K36 methylation in bovine cumulus cells

This study aims to investigate the expression and function of absent, small, or homeotic 1-like (ASH1L) methyltransferase in bovine cumulus cells in order to reveal by which mechanisms ASH1L regulates epigenetic modification and apoptosis in cumulus cells. The location of ASH1L and the methylation pattern of H3K36 were detected using immunofluorescence staining in cumulus cells. Quantitative PCR (qPCR) and western blotting were used to screen for effective siRNA targeting the ASH1L gene. Also, the mRNA expression levels of apoptosis-related genes and polycomb inhibitory complex genes were estimated by qPCR after knocking down the ASH1L gene in bovine cumulus cells. Cell proliferation and apoptosis were measured with the CCK-8 method and Annexin V-FITC by flow cytometry, respectively. The results of immunofluorescence analysis showed that ASH1L is located in the nucleus of bovine cumulus cells and is distributed in a dotted pattern. ASH1L knockdown in cumulus cells induced a decrease in the levels of H3K36me1/2/3 methylation (P < 0.05). Additionally, ASH1L knockdown inhibited cell proliferation, increased the apoptosis rate, and upregulated the expression of apoptosis genes CASPASE-3, BAX and BAX/BCL-2 ratio (P < 0.05). Meanwhile, the mRNA expression levels of EZH2 and SUZ12, two subunits of PRC2 protein, were increased in cells with ASH1L knockdown (P < 0.05). Therefore, the expression of ASH1L methyltransferase and its function in on the apoptosis of bovine cumulus cells were first studied. The mechanism by which ASH1L regulates the histone methylation and apoptosis in cumulus cells was also revealed.

Mechanism of Abnormal Chondrocyte Proliferation Induced by Piezo1-siRNA Exposed to Mechanical Stretch.

Objective To investigate the effect of small interfering RNA targeting mechanosensitive ion channel protein Piezo1 (Piezo1-siRNA) on abnormal chondrocyte proliferation exposed to mechanical stretch. Methods Construct and screen effective Piezo1-siRNA sequences and explore an appropriate method to transfect lentiviral vector into chondrocytes exposed to mechanical stretch. Western blot and RT-PCR were used to detect the mRNA and protein expression of Piezo1, Kif18A, and β-tubulin, respectively. Flow cytometry was used to measure the changes in the chondrocyte cycle. The proliferation of chondrocyte was evaluated by cell counting kit-8. Results According to the mRNA and protein expression of Piezo1, the effective siRNA sequence was successfully screened. Compared with the 0 h group, mechanical stretch upregulated the expression of Piezo1, Kif18A, and β-tubulin, resulting in chondrocyte cycle arrest and eventually inhibiting chondrocyte proliferation. Moreover, Piezo1-siRNA transfection effectively blocks this process and promotes the proliferation of chondrocyte. Conclusion Piezo1-siRNA can reduce the inhibition of chondrocyte proliferation induced by mechanical stretch via downregulating the expression of Kif18A and inhibiting the depolymerization of microtubules. Piezo1-siRNA plays a protective role in chondrocytes, which provides a potential method for the treatment of OA under abnormal mechanical stimulation.

Enzymatically synthesized 2′-fluoro-modified Dicer-substrate siRNA swarms against herpes simplex virus demonstrate enhanced antiviral efficacy and low cytotoxicity

Chemical modifications of small interfering (si)RNAs are used to enhance their stability and potency, and to reduce possible off-target effects, including immunogenicity. We have earlier introduced highly effective antiviral siRNA swarms against herpes simplex virus (HSV), targeting 653 bp of the essential UL29 viral gene. Here, we report a method for enzymatic production and antiviral use of 2′-fluoro-modified siRNA swarms. Utilizing the RNA-dependent RNA polymerase from bacteriophage phi6, we produced 2′-F-siRNA swarms containing either all or a fraction of modified adenosine, cytidine or uridine residues in the antisense strand of the UL29 target. The siRNA containing modified pyrimidines demonstrated high resistance to RNase A and the antiviral potency of all the UL29-specific 2′-F-siRNA swarms was 100-fold in comparison with the unmodified counterpart, without additional cytotoxicity. Modest stimulation of innate immunity signaling, including induced expression of both type I and type III interferons, as well as interferon-stimulated gene 54, by 2′-F-cytidine and 2′-F-uridine modified siRNA swarms occurred at early time points after transfection while the 2′-F-adenosine-containing siRNA was similar to the unmodified antiviral siRNA swarm in this respect. The antiviral efficacy of the 2′-F-siRNA swarms and the elicited cellular innate responses did not correlate suggesting that innate immunity pathways do not significantly contribute to the observed enhanced antiviral activity of the modified siRNAs. The results support further applications of enzymatically produced siRNA molecules with incorporated adenosine nucleotides, carrying fluoro-modification on ribose C2′ position, for further antiviral studies in vitro and in vivo.

Knockdown of Angiopoietin-like protein 4 suppresses the development of colorectal cancer

Colorectal cancer, is the growth of cancer cells in the part of the colon. Angiopeptin is one of the growth factors in the human body that is particularly effective in the regulatory process. In this research, the regulatory role and its mechanism of Angiopoietin-like protein 4 (ANGPTL4) in colorectal cancer (CRC) metastasis, has been studied. Protein expression of ANGPLT4 was analyzed by immunohistochemistry in tumor samples and adjacent normal specimens of 40 patients with CRC cancer of various phases. A gene knockout test was conducted, two effective siRNA of ANGPTL4, named siRNA1 and siRNA2, were constructed and transfected into two CRC cell lines SW480 and HT-29 to block the expression of ANGPTL4. QRT-PCR and western blotting were used to validate the knockdown efficiency of the mRNA and proteins. Based on the results, the protein expression of ANGPTL4 was increased in human CRC tissues with the development of CRC. Knockdown of ANGPTL4 by siRNA in SW480 and HT-29 cells in vitro inhibited cell proliferation, promoted cell apoptosis, and suppressed the ability of cell migration and invasion. Besides, the sensitivity of CRC cells to Cisplatin was increased in the low ANGPTL4 expression group. ANGPTL4 might be a new potential therapeutic target for patients with CRC.