Gene Silencing Research Articles

NQO1-Activatable Circular Antisense Oligonucleotides for Tumor-Cell-Specific Survivin Gene Silencing and Antitumor Therapy.

NAD(P)H:quinone oxidoreductase-1 (NQO1), a protein highly expressed in tumor cells, serves as an excellent trigger for releasing drugs specifically within tumor cells. In this study, we designed an activatable circular antisense oligonucleotide (cASO) by incorporating a head-to-tail cyclization mediated by an NQO1-responsive trimethyl-locked quinone propionate (Q3PA), coupled with a self-immolative linker. The resulting circular structure prevented the cASO from binding to the target mRNA, thereby avoiding gene silencing. However, upon encountering NQO1, the circular form was converted to a linear form, leading to the silencing of the targeted gene. In vitro experiments demonstrated significant tumor-cell-specific activity of the cASO, while in vivo studies using an A549-Luc orthotopic lung tumor model revealed a substantial antitumor effect, primarily attributed to the suppression of survivin expression. This NQO1-activatable cASO represents a novel strategy for achieving tumor-cell-specific gene silencing and holds promise for the development of ASO prodrugs with enhanced therapeutic potentials.

Beyond species and spatial boundaries: Enabling long-distance gene silencing in plants via guanidinium-siRNA nanoparticles.

RNA interference (RNAi) has been widely used in agriculture. However, it is well accepted that common methods of plant RNAi are species-dependent and lack systematic efficiency. This study designed a thiolated siRNA nanoparticle, guanidinium (Gu+)-containing disulfide assembled siRNA (Gu+-siRNA), demonstrating remarkable species independence and efficient systemic gene silencing across different plant species. Our results indicate that this approach effectively utilizes the plant vascular system to deliver siRNA, enabling long-distance gene silencing across both monocot and dicot plants, such as rice and Arabidopsis. By applying this method, we successfully targeted and silenced key genes like STM, WER, MYB23, GD1, EIL1, and EIL2, which regulate plant development and enhance salt tolerance. This delivery system significantly expands the application of RNAi technology across different plants, serving as a valuable tool for advancing agricultural biotechnology, enhancing crop resistance, and improving agricultural productivity, while aligning with global goals for sustainable food production and crop improvement.

Spray-induced gene silencing to control plant pathogenic fungi: A step-by-step guide.

RNA interference (RNAi)-based control technologies are gaining popularity as potential alternatives to synthetic fungicides in the ongoing effort to manage plant pathogenic fungi. Among these methods, spray-induced gene silencing (SIGS) emerges as particularly promising due to its convenience and feasibility for development. This approach is a new technology for plant disease management, in which double-stranded RNAs (dsRNAs) targeting essential or virulence genes are applied to plants or plant products and subsequently absorbed by plant pathogens, triggering a gene silencing effect and the inhibition of the infection process. Spray-induced gene silencing has demonstrated efficacy in laboratory settings against various fungal pathogens. However, as research progressed from the laboratory to the greenhouse and field environments, novel challenges arose, such as ensuring the stability of dsRNAs and their effective delivery to fungal targets. Here, we provide a practical guide to SIGS for the control of plant pathogenic fungi. This guide outlines the essential steps and considerations needed for designing and assessing dsRNA molecules. It also addresses key challenges inherent to SIGS, including delivery and stability of dsRNA molecules, and how nanoencapsulation of dsRNAs can aid in overcoming these obstacles. Additionally, the guide underscores existing knowledge gaps that warrant further research and aims to provide assistance to researchers, especially those new to the field, encouraging the advancement of SIGS for the control of a broad range of fungal pathogens.

The Silencing of the StPAM16-1 Gene Enhanced the Resistance of Potato Plants to the Phytotoxin Thaxtomin A

The Silencing of the StPAM16-1 Gene Enhanced the Resistance of Potato Plants to the Phytotoxin Thaxtomin A

Remote Regulation by VirB, the Transcriptional Anti-Silencer of Shigella Virulence Genes, Provides Mechanistic Information.

Classical models of bacterial transcription show regulators binding close to promoter elements to exert their effect. However, the scope for long-range regulation exists, especially by nucleoid structuring proteins, like H-NS. Here, long-range regulation by VirB, a transcriptional regulator that alleviates H-NS-mediated silencing of key virulence genes in Shigella species, is explored invivo to test the limits of long-range regulation and provide further mechanistic insight. VirB-dependent regulation of the well-characterized icsP promoter persists if its cognate site is repositioned 1 kb, 3.3 kb, and even 4.7 kb further upstream than its native position in a plasmid reporter. VirB-dependent regulation diminishes with binding site distance. While increasing cellular VirB pools elevated promoter activity in all constructs with wild-type VirB binding sites, it did not generate a disproportionate increase in promoter activity from remote sites relative to the native site. Since VirB occludes a constitutively active promoter (PT5) when docked adjacent to its -35 element, we next moved the VirB binding site far outside the promoter region. We discovered that VirB still interfered with promoter activity. These findings and those generated from molecular roadblocks engineered around a distally located VirB-binding site are reconciled with the various models of transcriptional regulation by VirB.

Role of spinal Barrier-to-Autointegration Factor (BAF) in the epigenetic silencing of the mu-opioid receptor gene in neuropathic pain

BackgroundNeuropathic pain presents a significant clinical challenge, with spinal cord epigenetic mechanisms playing a critical role in its development. This study investigated the impact of nerve injury on the Barrier-to-Autointegration...

Cell-Penetrating Peptide-Mediated Delivery of Gene-Silencing Nucleic Acids to the Invasive Common Reed Phragmites australis via Foliar Application

As a popular tool for gene function characterization and gene therapy, RNA interference (RNAi)-based gene silencing has been increasingly explored for potential applications to control invasive species. At least two major hurdles exist when applying this approach to invasive plants: (1) the design and screening of species- and gene-specific biomacromolecules (i.e., gene-silencing agents or GSAs) made of DNA, RNA, or peptides that can suppress the expression of target genes efficiently, and (2) the delivery vehicle needed to penetrate plant cell walls and other physical barriers (e.g., leaf cuticles). In this study, we investigated the cell-penetrating peptide (CPP)-mediated delivery of multiple types of GSAs (e.g., double-stranded RNA (dsRNA), artificial microRNA (amiRNA), and antisense oligonucleotide (ASO)) to knock down a putative phytoene desaturase (PDS) gene in the invasive common reed (Phragmites australis spp. australis). Both microscopic and quantitative gene expression evidence demonstrated the CPP-mediated internalization of GSA cargos and transient suppression of PDS expression in both treated and systemic leaves up to 7 days post foliar application. Although various GSA combinations and application rates and frequencies were tested, we observed limitations, including low gene-silencing efficiency and a lack of physiological trait alteration, likely owing to low CPP payload capacity and the incomplete characterization of the PDS-coding genes (e.g., the recent discovery of two PDS paralogs) in P. australis. Our work lays a foundation to support further research toward the development of convenient, cost-effective, field-deployable, and environmentally benign gene-silencing technologies for invasive P. australis management.

PbMADS49 Regulates Lignification During Stone Cell Development in 'Dangshansuli' (Pyrus bretschneideri) Fruit.

Lignified stone cell content is one of the critical factors affecting 'Dangshansuli' fruit quality. The function of MADS-box transcription factors in regulating lignin biosynthesis in pear fruit is still less. In this study, PbMADS49 gene silencing inhibited the lignin biosynthesis and stone cell secondary wall development of pear fruit mainly through reducing the expression levels of lignin monomer polymerisation key enzymes (PbPRX33 and PbPRX45). PbMADS49 was a transcriptional repressor inhibiting its transcription by binding to the CArG element in the target gene promoter. Combined with the co-expression network and promoter cis-acting element analysis, we hypothesised that PbMADS49 positively regulates the transcription of PbPRX33 through PbWRKY63. The gene silencing effect of homologous genes PbPRX33-1 and PbPRX33-2 was consistent with PbMADS49, and PbPRX33-2 was more significant than PbPRX33-1. This study shows that PbMADS49 is a positive regulator of stone cell lignification, providing new insights into the development mechanism of pear stone cells.

Identification of mitochondrial carrier homolog 2 as an important therapeutic target of castration-resistant prostate cancer

We here investigate the expression of the mitochondrial carrier homolog 2 (MTCH2) and its potential function in castration-resistant prostate cancer (CRPC). Bioinformatic analyses reveal that MTCH2 overexpression is associated with critical clinical parameters of prostate cancer. Single-cell sequencing data indicate elevated MTCH2 expression in the prostate cancer epithelium. MTCH2 is also upregulated in locally treated CRPC tissue and various primary human CRPC cells. Using genetic silencing via shRNA and knockout (KO) through the CRISPR-sgRNA approach, we showed that the depletion of MTCH2 impaired mitochondrial function, resulting in a reduced oxygen consumption rate, diminished complex I activity, and decreased ATP levels, mitochondrial depolarization, and increased reactive oxygen species production in primary CRPC cells. The silencing or KO of MTCH2 significantly inhibited cell viability, proliferation, and migration, together with a marked increase in apoptosis in the primary CRPC cells. In contrast, ectopic expression of MTCH2 provided CRPC cells with pro-tumorigenic properties, enhancing ATP production and promoting cell proliferation and migration. MTCH2 silencing also markedly inhibited the growth of subcutaneous xenografts of the primary CRPC cells in nude mice. The MTCH2-silenced xenografts exhibited increased apoptosis, elevated lipid peroxidation, and decreased ATP levels. These results provide new insights into the role of MTCH2 in supporting mitochondrial function and CRPC progression.

Fatty Acid ABCG Transporter GhSTR1 Mediates Resistance to Verticillium dahliae and Fusarium oxysporum in Cotton

Verticillium wilt and Fusarium wilt cause significant losses in cotton (Gossypium hirsutum) production and have a significant economic impact. This study determined the functional role of GhSTR1, a member of the ABCG subfamily of ATP-binding cassette (ABC) transporters, that mediates cotton defense responses against various plant pathogens. We identified GhSTR1 as a homolog of STR1 from Medicago truncatula and highlighted its evolutionary conservation and potential role in plant defense mechanisms. Expression profiling revealed that GhSTR1 displays tissue-specific and spatiotemporal dynamics under stress conditions caused by Verticillium dahliae and Fusarium oxysporum. Functional validation using virus-induced gene silencing (VIGS) showed that silencing GhSTR1 improved disease resistance, resulting in milder symptoms, less vascular browning, and reduced fungal growth. Furthermore, the AtSTR1 loss-of-function mutant in Arabidopsis thaliana exhibited similar resistance phenotypes, highlighting the conserved regulatory role of STR1 in pathogen defense. In addition to its role in disease resistance, the mutation of AtSTR1 in Arabidopsis also enhanced the vegetative and reproductive growth of the plant, including increased root length, rosette leaf number, and plant height without compromising drought tolerance. These findings suggest that GhSTR1 mediates a trade-off between defense and growth, offering a potential target for optimizing both traits for crop improvement. This study identifies GhSTR1 as a key regulator of plant–pathogen interactions and growth dynamics, providing a foundation for developing durable strategies to enhance cotton’s resistance and yield under biotic and abiotic stress conditions.

Role of Atg3, Atg5 and Atg12 in the crosstalk between apoptosis and autophagy in the posterior silk gland of Bombyx mori.

Autophagy is a cellular mechanism that enhances cell survival in response to various stressors, including nutrient deprivation; however, it also plays a pivotal role in the regulation of programmed cell death. This study examined the effects of autophagy-related genes Atg3, Atg5 and Atg12 on apoptosis and autophagy during the degeneration of the posterior silk gland in Bombyx mori, employing RNA interference techniques. Apoptosis-specific markers and autophagic processes were evaluated in both control and treatment groups. The knockdown of all three genes resulted in a significant reduction in autophagy, modifications in the apoptosis process, aberrant expression of p53 and impaired lysosomal function. It was determined that Atg3 is involved in the regulation of intracellular mitochondrial homeostasis. Following the silencing of Atg5, evidence was obtained indicating the gene's role in regulating lysosomal pH. Notably, the loss of Atg3 and Atg5 was associated with an increase in apoptotic markers, whereas the silencing of Atg12 inhibited apoptosis. Elevated levels of the p53 transcription factor following gene silencing suggested a potential interaction between these genes and p53. Our findings further underscore the importance of autophagy-mediated cell death, involving Atg3, Atg5 and Atg12, in the proper progression of degeneration in the posterior silk gland. A comprehensive understanding of the molecular mechanisms that mediate the interaction between apoptosis and autophagy is essential for elucidating their roles in both physiological and pathological contexts.

Transcription Factor RhCUC3 Regulates Petal Numbers in Rose Flowers

Rose is one of the most popular ornamental plants worldwide. The double-flower trait, referring to flowers with extra petals, has been a key focus in rose breeding history. However, the genetic mechanisms regulating petal number in roses are still not fully understood. Here, we identified the CUP-SHAPED COTYLEDON 3 (RhCUC3) gene in the miniature rose (Rosa hybrida ‘Eclair’). The expression of RhCUC3 was high during the petal and stamen primordium differentiation stages but declined sharply during pistil primordium development. RhCUC3 belongs to the NAM/CUC3 subgroup of NAC transcription factors and is localized in the nucleus. The transcript level of RhCUC3 increased significantly with ABA and GA treatments and was inversely down-regulated with MeJA and 6-BA treatments. Silencing RhCUC3 using virus-induced gene silencing (VIGS) in rose ‘Eclair’ significantly decreased the number of petaloid stamens and normal petals while slightly increasing the number of stamens. Additionally, the expression of RhAG and RhAGL, two MADS-box genes associated with floral organ identity, was significantly higher in TRV-RhCUC3 compared to the TRV control. These findings suggest that RhCUC3 enhances stamen petaloidy and petal number, potentially by modulating the expression of RhAG and RhAGL, providing new insights into the function of NAC transcription factors in plants.

Synthesis of Composites of DNA and Boron Cluster‐Metal Complexes and their Assembly into Functional Nanoparticles with Gene Silencing Activity

AbstractHerein, we describe a new type of building block for nanoconstruction based on the composite of a boron cluster metal‐complex (metallacarborane) and therapeutic nucleic acid and their assembly into functional nanoparticles with EGFR oncogene silencing activity. In this direction, a method for oligofunctionalization of a metallacarborane, 3,3’‐cobalt‐bis(1,2‐dicarbollide)(−1) ion, and an automated method for synthesis of DNA‐oligonucleotides attached to the metallacarborane core is developed. The obtained building blocks forms nanoparticles with a “chrysanthemum‐like” topology that carry a slowly releasing antisense anti‐EGFR oligonucleotide silencing EGFR gene efficiently in vitro in a pancreatic carcinoma cell line PANC‐1.

Mechanistic insights into 5-aminolevulinic acid photodynamic therapy for acne vulgaris: targeting lipogenesis via the OLR1-Wnt/β-catenin pathway

Acne vulgaris, a prevalent chronic inflammatory skin disorder, is often characterized by hyperactive sebaceous glands and excessive sebum production, presenting a significant therapeutic challenge. While 5-aminolevulinic acid photodynamic therapy (ALA-PDT) is clinically effective in treating moderate to severe acne, the molecular mechanisms underlying its therapeutic effects remain largely unexplored. In this study, we investigated the impact of ALA-PDT on lipid metabolism in an acne-like mouse model and in immortalized human sebocytes (XL-i-20), focusing on the role of the OLR1-Wnt/β-catenin pathway. We employed transcriptomic analysis, lipid staining, and gene silencing techniques to dissect the molecular interactions induced by ALA-PDT. Our findings revealed that ALA-PDT significantly reduces lipogenesis by upregulating OLR1, which in turn suppresses the SREBP1-FAS axis, thereby decreasing lipid accumulation in sebocytes. Furthermore, activation of the OLR1-Wnt/β-catenin pathway was essential for these lipogenic effects, as silencing OLR1 or activating Wnt/β-catenin signaling reversed lipogenesis inhibition. This study elucidates a novel mechanistic pathway in ALA-PDT-mediated acne treatment, highlighting OLR1 as a promising target for future therapeutic strategies.

Hypothalamic Estrogen Receptor α Is Essential for Female Marmoset Sexual Behavior Without Protecting From Obesity.

Estrogen receptor α (ERα) in the ventromedial (VMN) and arcuate (ARC) nuclei of female rodent mediobasal hypothalami (MBHs) provides a crucial molecular gateway facilitating estradiol (E2) regulation of sexual behavior, reproductive neuroendocrinology, and metabolic function. In female nonhuman primates (NHPs) and women, however, its hypothalamic counterpart remains unknown. We hypothesized that knockdown (KD) of ERα expression in the hypothalamic VMN and ARC of female marmosets would diminish sexual receptivity, while simultaneously disrupting gonadotropic and metabolic homeostasis. We ovariectomized (OVX) adult female marmosets of comparable age and weight, immediately replaced E2 at midcycle levels, and approximately 1 month later assigned monkeys to diet-induced obesity (DIO) within group (1) control, receiving scrambled short hairpin RNA (shRNA), or (2) ERαKD, receiving selective ERα gene silencing shRNA. Magnetic resonance imaging-guided neural surgery enabled hypothalamic infusion of viral vector shRNA and subsequent brain immunohistochemistry enabled observer-validated, NIS-elements computer software quantification of ERα knockdown. ERα expression was significantly diminished in the VMN and ARC, but not the preoptic area (POA), of ERαKD females coincident with elimination of timely female sexual responses, more than 80% loss of female receptivity, modestly elevated gonadotropin levels, hyperglycemia, and diminished calorie consumption. Density and intensity of ERα-expressing cells in the VMN correlated positively with female sexual receptivity and calorie consumption, negatively with timeliness of female sexual responses, and in the ARC, correlated negatively with calorie consumption. ERα activation in the female NHP MBH is critically important for female sexual behavior and modestly contributes to gonadotropic and metabolic control.

Pericytes Are Odontoblast Progenitor Cells Depending on ER Stress.

Odontoblasts are terminally differentiated cells that exhibit mechanosensitivity and mineralization capacity. Mechanosensitive ion channels such as Piezo1 are present in odontoblasts and are associated with their physiological functions via Ca2+ signaling. Both Ca2+ signals via Ca2+ influx from mechanosensitive ion channels and Ca2+ release from Ca2+ stores function as secondary messenger systems for various biological phenomena. The endoplasmic reticulum (ER) serves as an intracellular Ca2+ store that mobilizes intracellular Ca2+. Changes in Ca2+ concentration inside the ER are among the factors that cause ER stress. Perivascular cells are located around odontoblasts in the dental pulp. Although such formation indicates that perivascular cells interact with odontoblasts, their detailed profiles under developmental and pathological conditions remain unclear. In this study, we revealed that pericyte marker, neural/glial antigen 2 (NG2)-positive cells, in cell-rich zones (CZs) can differentiate into Piezo1-positive odontoblasts following genetic odontoblast depletion in mice, and modeled as odontoblast death after severe dentin injury and as reparative dentin formation. NG2-positive pericytes differentiated into odontoblasts faster than glial cells. To determine how NG2-positive cells differentiate into Piezo1-positive odontoblasts, we focused on the ER-stress sensor protein, activating transcription factor 6a (ATF6a). After genetic odontoblast depletion, NG2-positive cells regenerated in the odontoblast layer and were capable of acting as functional odontoblasts. In the presence of extracellular Ca2+, the application of a sarco/ER Ca2+-ATPase (SERCA) inhibitor, thapsigargin, known as an ER-stress inducer, increased the intracellular Ca2+ concentration in the odontoblast lineage cells (OLCs). The increase was significantly inhibited by the application of a pharmacologic Piezo1 inhibitor, indicating that ER stress by SERCA inhibition augmented Piezo1-induced responses in odontoblast progenitor cells. However, the physiological activation of Gq-coupled receptors by adenosine diphosphate did not induce Piezo1 activation. Gene silencing of ATF6a and/or NG2 impaired the mineralization of OLCs. Overall, ATF6a orchestrates the differentiation of NG2-positive pericytes into functional odontoblasts that act as sensory receptor cells and dentin-forming cells.

Intergenerational transport of double-stranded RNA in C. elegans can limit heritable epigenetic changes.

RNAs in circulation carry sequence-specific regulatory information between cells in plant, animal, and host-pathogen systems. Such RNA can cross generational boundaries, as evidenced by somatic double-stranded RNA (dsRNA) in the nematode Caenorhabditis elegans silencing genes of matching sequence in progeny. Here we dissect the intergenerational path taken by dsRNA from parental circulation and discover that cytosolic import through the dsRNA importer SID-1 in the parental germline and/or developing progeny varies with developmental time and dsRNA substrates. Loss of SID-1 enhances initiation of heritable RNA silencing within the germline and causes changes in the expression of the sid-1-dependent gene sdg-1 that last for more than 100 generations after restoration of SID-1. The SDG-1 protein is enriched in perinuclear germ granules required for heritable RNA silencing but is expressed from a retrotransposon targeted by such silencing. This auto-inhibitory loop suggests how retrotransposons could persist by hosting genes that regulate their own silencing.

Intergenerational transport of double-stranded RNA in C. elegans can limit heritable epigenetic changes

RNAs in circulation carry sequence-specific regulatory information between cells in plant, animal, and host-pathogen systems. Such RNA can cross generational boundaries, as evidenced by somatic double-stranded RNA (dsRNA) in the nematode Caenorhabditis elegans silencing genes of matching sequence in progeny. Here we dissect the intergenerational path taken by dsRNA from parental circulation and discover that cytosolic import through the dsRNA importer SID-1 in the parental germline and/or developing progeny varies with developmental time and dsRNA substrates. Loss of SID-1 enhances initiation of heritable RNA silencing within the germline and causes changes in the expression of the sid-1-dependent gene sdg-1 that last for more than 100 generations after restoration of SID-1. The SDG-1 protein is enriched in perinuclear germ granules required for heritable RNA silencing but is expressed from a retrotransposon targeted by such silencing. This auto-inhibitory loop suggests how retrotransposons could persist by hosting genes that regulate their own silencing.

Characterization of Rationally Designed CRISPR/Cas9-Based DNA Methyltransferases with Distinct Methyltransferase and Gene Silencing Activities in Human Cell Lines and Primary Human T Cells.

Nuclease-deactivated Cas (dCas) proteins can be used to recruit epigenetic effectors, and this class of epigenetic editing technologies has revolutionized the ability to synthetically control the mammalian epigenome and transcriptome. DNA methylation is one of the most important and well-characterized epigenetic modifications in mammals, and while many different forms of dCas-based DNA methyltransferases (dCas-DNMTs) have been developed for programmable DNA methylation, these tools are frequently poorly tolerated and/or lowly expressed in mammalian cell types. Further, the use of dCas-DNMTs has largely been restricted to cell lines, which limits mechanistic insights in karyotypically normal contexts and hampers translational utility in the longer term. Here, we extend previous insights into the rational design of the catalytic core of the mammalian DNMT3A methyltransferase and test three dCas9-DNMT3A/3L variants across different human cell lines and in primary donor-derived human T cells. We find that mutations within the catalytic core of DNMT3A stabilize the expression of dCas9-DNMT3A/3L fusion proteins in Jurkat T cells without sacrificing DNA methylation or gene-silencing performance. We also show that these rationally engineered mutations in DNMT3A alter DNA methylation profiles at loci targeted with dCas9-DNMT3A/3L in cell lines and donor-derived human T cells. Finally, we leverage the transcriptionally repressive effects of dCas9-DNMT3A/3L variants to functionally link the expression of a key immunomodulatory transcription factor to cytokine secretion in donor-derived T cells. Overall, our work expands the synthetic biology toolkit for epigenetic editing and provides a roadmap for the use of engineered dCas-based DNMTs in primary mammalian cell types.

Microscopy methods for the in vivo study of nanoscale nuclear organization.

Eukaryotic genomes are highly compacted within the nucleus and organized into complex 3D structures across various genomic and physical scales. Organization within the nucleus plays a key role in gene regulation, both facilitating regulatory interactions to promote transcription while also enabling the silencing of other genes. Despite the functional importance of genome organization in determining cell identity and function, investigating nuclear organization across this wide range of physical scales has been challenging. Microscopy provides the opportunity for direct visualization of nuclear structures and has pioneered key discoveries in this field. Nonetheless, visualization of nanoscale structures within the nucleus, such as nucleosomes and chromatin loops, requires super-resolution imaging to go beyond the ~220 nm diffraction limit. Here, we review recent advances in imaging technology and their promise to uncover new insights into the organization of the nucleus at the nanoscale. We discuss different imaging modalities and how they have been applied to the nucleus, with a focus on super-resolution light microscopy and its application to in vivo systems. Finally, we conclude with our perspective on how continued technical innovations in super-resolution imaging in the nucleus will advance our understanding of genome structure and function.