Gene Silencing Research Articles

Foxa deficiency restricts hepatitis B virus biosynthesis through epigenic silencing.

The current absence of curative therapies capable of resolving chronic hepatis B virus (HBV) infection is a major clinical problem associated with considerable morbidity and mortality. The small viral genome limits molecular targets for drug development, suggesting that the identification of cellular factors essential for HBV biosynthesis may represent alternative targets for therapeutic intervention. Genetic Foxa deficiency in the neonatal liver of HBV transgenic mice leads to the transcriptional silencing of viral DNA by CpG methylation without affecting viability or displaying an obvious phenotype. Therefore, limiting liver Foxa activity therapeutically may lead to the methylation of viral covalently closed circular DNA (cccDNA), resulting in its transcriptional silencing and ultimately the resolution of chronic HBV infection.

Revolutionizing Cardiac Care: The Role of Gene Therapy in Treating Cardiomyopathy

Gene therapy presents a method for addressing types of cardiomyopathies that play a substantial role in heart failure. This innovative approach, leveraging technologies such as clustered regularly interspaced short palindromic repeats/Cas9 for modifying genomes, holds promise for lasting treatments or potential cures that go beyond therapies. It is essential to grasp the workings of gene therapy, including gene silencing, clustered regularly interspaced short palindromic repeats genome editing, and enhancing sarcomere function to effectively apply it to treating cardiomyopathy. Examining current trials will shed light on the advancements and accomplishments in this field while also addressing the obstacles, uncertainties, and opportunities ahead. Delving into the possibilities of gene therapy involves exploring targets and inventive delivery methods that underscore the evolving landscape of research in this domain hinting at a future brimming with opportunities to transform care. The progress made in using gene therapy to treat cardiomyopathies represents the progress of medicine in driving forward scientific innovation to provide more precise and enduring solutions for patients. Continuously refining gene therapy techniques and deepening our knowledge of genetics are factors that will shape the future direction of cardiac care. The potential of gene therapy does not just benefit individuals with cardiomyopathy but also represents a move toward effective treatments for various genetic conditions. This signifies a step in the pursuit of holistic healthcare solutions.

A novel gene silencing strategy based on tobacco rattle virus in Hibiscus mutabilis.

Hibiscus mutabilis L. is a popular regional characteristic plant in China, cultivated for its attractive flower colors, extended bloom time, and medicinal properties. To enhance molecular breeding and gene function studies, we conducted transcriptome analysis and identified valuable genes in previous research. Nonetheless, the current inefficient and labor-intensive transformation techniques have hindered their applications. Virus-induced gene silencing (VIGS) provides a precise and effective strategy for post-transcriptional down-regulation of endogenous gene expression. We investigated the performance of tobacco rattle virus (TRV) as a tool for targeting and silencing the gene encoding the protein involved in chloroplast development, cloroplastos alterados 1 (altered chloroplast; CLA1), of H. mutabilis through Agrobacterium tumefaciens-mediated infiltration. By effectively suppressing the CLA1 gene associated with chloroplast development in H. mutabilis via the TRV-VIGS system, we have illustrated the inaugural implementation of VIGS in this species. Quantitative RT-PCR proved that HmCLA1 expression in agro-infiltrated plants was lower than in the mock-infiltrated (mock) and the control (CK) plants. Phenotypic observations corroborated the albino phenotype in leaves following successful HmCLA1 silencing. Our study showcases TRV-VIGS as a potential gene silencing tool for H. mutabilis, facilitating functional genomics studies and molecular breeding efforts in this species.

The HSV-1 encoded CCCTC-binding factor, CTRL2, impacts the nature of viral chromatin during HSV-1 lytic infection.

HSV-1 genomes are rapidly heterochromatinized following entry by host cells to limit viral gene expression. Efficient HSV-1 genome replication requires mechanisms that de-repress chromatin associated with the viral genome. CCCTC-binding factors, or CTCF insulators play both silencing and activating roles in cellular transcriptional regulation. Importantly, the HSV-1 genome encodes several CTCF insulators that flank IE genes, implying that individual HSV-1 encoded CTCF insulators regulate IE transcription during all stages of the HSV-1 life cycle. We previously reported that the HSV-1 encoded CTCF insulator located downstream of the LAT (CTRL2) controlled IE gene silencing during latency. To further characterize the role of this insulator during the lytic infection we leveraged a ΔCTRL2 recombinant virus to show that there was a genome replication defect that stemmed from decreased IE gene expression in fibroblasts and epithelial cells at early times following initiation of infection. Further experiments indicated that the defect in gene expression resulted from chromatin inaccessibility in the absence of the insulator. To elucidate how chromatin accessibility was altered in the absence of the CTRL2 insulator, we showed that enrichment of Alpha-thalassemia/mental retardation, X-linked chromatin remodeler (ATRX), and the histone variant H3.3, both of which are known for their roles in maintaining repressive histone markers on the HSV-1 viral genome were increased on IE regions of HSV-1. Finally, both H3K27me3 and H3K9me3 repressive histone marks remained enriched by 4 hours post infection in the absence of the CTRL2 insulator, confirming that the CTRL2 insulator is required for de-repression of IE genes of viral genomes. To our knowledge these are the first data that show that a specific CTCF insulator in the HSV-1 genome (CTRL2) regulates chromatin accessibility during the lytic infection.

Female-bias in systemic lupus erythematosus: How much is the X chromosome to blame?

Systemic lupus erythematosus (SLE or lupus) is an immune-mediated disease associated with substantial medical burden. Notably, lupus exhibits a striking female bias, with women having significantly higher susceptibility compared to men, up to 14-fold higher in some ethnicities. Supernumerary X chromosome syndromes, like Klinefelter (XXY) and Triple X syndrome (XXX), also present higher SLE prevalence, whereas Turner syndrome (XO) displays lower prevalence. Taken together, SLE prevalence in different X chromosome dosage sceneries denotes a relationship between the number of X chromosomes and the risk of developing lupus. The dosage of X-linked genes, many of which play roles in the immune system, is compensated between males and females through the inactivation of one of the two X chromosomes in female cells. X-chromosome inactivation (XCI) initiates early in development with a random selection of which X chromosome to inactivate, a choice that is then epigenetically maintained in the daughter cells. This process is regulated by the X-Inactive-Specific Transcript (XIST), encoding for a long non-coding RNA, exclusively expressed from the inactive X chromosome (Xi). XIST interacts with various RNA binding proteins and chromatin modifiers to form a ribonucleoprotein (RNP) complex responsible for the transcriptional silencing and heterochromatinization of the Xi. This ensures stable silencing of most genes on the X chromosome, with only a few genes able to escape this process. Recent findings suggest that the molecular components involved in XCI, or their dysregulation, contribute to the pathogenesis of lupus. Indeed, nonrandom XCI, elevated gene escape from XCI, and the autoimmune potential of the XIST RNP complex have been suggested to contribute to auto-immune diseases, such as lupus. This review examines these current hypotheses concerning how this dosage compensation mechanism might impact the development of lupus, shedding light on potential mechanisms underlying the pathogenesis of the disease.

Effect of Degree of Substitution and Molecular Weight on Transfection Efficacy of Starch-Based siRNA Delivery System

RNA interference (RNAi) is a promising approach for gene therapy in cancers, but it requires carriers to protect and deliver therapeutic small interfering RNA (siRNA) molecules to cancerous cells. Starch-based carriers, such as quaternized starch (Q-Starch), have been shown to be biocompatible and are able to form nanocomplexes with siRNA, but significant electrostatic interactions between the carrier and siRNA prevent its release at the target site. In this study, we aim to characterize the effects of the degree of substitution (DS) and molecular weight (Mw) of Q-Starch on the gene silencing capabilities of the Q-Starch/siRNA transfection system. We show that reducing the DS reduces the electrostatic interactions between Q-Starch and siRNA, which now decomplex at more physiologically relevant conditions, but also affects additional parameters such as complex size while mostly maintaining cellular uptake capabilities. Notably, reducing the DS renders Q-Starch more susceptible to enzymatic degradation by α-amylase during the initial Q-Starch pretreatment. Enzymatic cleavage leads to a reduction in the Mw of Q-Starch, resulting in a 25% enhancement in its transfection capabilities. This study provides a better understanding of the effects of the DS and Mw on the polysaccharide-based siRNA delivery system and indicates that the polysaccharide Mw may be the key factor in determining the transfection efficacy of this system.

Self-assembly and condensation of intermolecular poly(UG) RNA quadruplexes.

Poly(UG) or 'pUG' dinucleotide repeats are highly abundant sequences in eukaryotic RNAs. In Caenorhabditis elegans, pUGs are added to RNA 3' ends to direct gene silencing within Mutator foci, a germ granule condensate. Here, we show that pUG RNAs efficiently self-assemble into gel condensates through quadruplex (G4) interactions. Short pUG sequences form right-handed intermolecular G4s (pUG G4s), while longer pUGs form left-handed intramolecular G4s (pUG folds). We determined a 1.05 Å crystal structure of an intermolecular pUG G4, which reveals an eight stranded G4 dimer involving 48 nucleotides, 7 different G and U quartet conformations, 7 coordinated potassium ions, 8 sodium ions and a buried water molecule. A comparison of the intermolecular pUG G4 and intramolecular pUG fold structures provides insights into the molecular basis for G4 handedness and illustrates how a simple dinucleotide repeat sequence can form complex structures with diverse topologies.

Evolutionary comparison of lncRNAs in four cotton species and functional identification of LncR4682-PAS2-KCS19 module in fiber elongation.

Long non-coding RNAs (lncRNAs) play an important role in various biological processes in plants. However, there have been few reports on the evolutionary signatures of lncRNAs in closely related cotton species. The lncRNA transcription patterns in two tetraploid cotton species and their putative diploid ancestors were compared in this paper. By performing deep RNA sequencing, we identified 280 429 lncRNAs from 21 tissues in four cotton species. lncRNA transcription evolves more rapidly than mRNAs, and exhibits more severe turnover phenomenon in diploid species compared to that in tetraploid species. Evolutionarily conserved lncRNAs exhibit higher expression levels, and lower tissue specificity compared with species-specific lncRNAs. Remarkably, tissue expression of homologous lncRNAs in Gossypium hirsutum and G. barbadense exhibited similar patterns, suggesting that these lncRNAs may be functionally conserved and selectively maintained during domestication. An orthologous lncRNA, lncR4682, was identified and validated in fibers of G. hirsutum and G. barbadense with the highest conservatism and expression abundance. Through virus-induced gene silencing in upland cotton, we found that lncR4682 and its target genes GHPAS2 and GHKCS19 positively regulated fiber elongation. In summary, the present study provides a systematic analysis of lncRNAs in four closely related cotton species, extending the understanding of transcriptional conservation of lncRNAs across cotton species. In addition, LncR4682-PAS2-KCS19 contributes to cotton fiber elongation by participating in the biosynthesis of very long-chain fatty acids.

Single-Stranded Hairpin Loop RNAs (loopmeRNAs) Potently Induce Gene Silencing through the RNA Interference Pathway.

Synthetic small interfering RNAs conjugated to trivalent N-acetylgalactosamine (GalNAc) are clinically validated drugs for treatment of liver diseases. Incorporation of phosphorothioate linkages and ribose modifications are necessary for stability, potency, and duration of pharmacology. Although multiple alternative siRNA designs such as Dicer-substrate RNA, shRNA, and circular RNA have been evaluated in vitro and in preclinical studies with some success, clinical applications of these designs are limited as it is difficult to incorporate chemical modifications in these designs. An alternative siRNA design that can incorporate chemical modifications through straightforward synthesis without compromising potency will significantly advance the field. Here, we report a facile synthesis of GalNAc ligand-containing single-stranded loop hairpin RNAs (loopmeRNAs) with clinically relevant chemical modifications. We evaluated the efficiency of novel loopmeRNA designs in vivo and correlated their structure-activity relationship with the support of in vitro metabolism data. Sequences and chemical modifications in the loop region of the loopmeRNA design were optimized for maximal potency. Our studies demonstrate that loopmeRNAs can efficiently silence expression of target genes with comparable efficacy to conventional double-stranded siRNAs but reduced environmental and regulatory burdens.

6455 The Effect of Gene Silencing on Inhibiting Stress Hormones in Wild Birds Raised Under Stressful Conditions

Abstract Disclosure: S.M. Abdulateef: None. T. Mohammed: None. W. Al-Jugif: None. M.Q. Abed: None. Wild birds are notable for their ease of rearing, tolerance to extreme environmental conditions such as heat and cold, and their resistance to diseases. They are also recognized for their ability to withstand stress. However, their primary challenge is low egg production, stemming from their aggressive nature. This aggression elevates the secretion of stress and fear hormones, significantly impacting reproductive hormones. This study aims to explore the effects of gene silencing on the physiological traits of wild birds, particularly its influence on physiological responses and the reduction of stress hormone levels. Three treatments were employed: T1 (control), T2 (welfare treatment, wherein birds were raised with enhanced well-being), and T3 (using siRNA technology to silence the gene responsible for stress hormone corticosterone production). The silencing of CYP11B1 and CYP11B2 involved obtaining the cDNA of each gene from the NCBI, associated with corticosterone production. The coding sequence from the 5' end at the cap direction was reversed to complement mRNA. Two siRNAs were procured from Macrogene Company (South Korea) in lyophilized form at high concentration, with sequences: CYP11B2 5'-acacctctgcctttgccctgagtgccat-3' and CYP11B1 5'-ctaaagtcaaactcagccccacattcat-3'. These were dissolved in molecular-grade distilled water for a final concentration of 500 pmol/µl. Each hen was injected in the wing in the jugular artery with 125 µl of the siRNA solution at 14 and 18 weeks of age, before the commencement of the egg-laying cycle. Corticosterone levels were measured using the Sunlong Biotech CO., LTD kit. The results showed a significant decrease (p<0.01) in stress hormone levels in the third treatment group (gene silencing treatment), leading to notable physiological responses in the birds compared to the other treatments. This study highlights the impact of gene silencing on the physiological traits of wild birds and its effects on their physiological responses. The findings of this study are significant in the context of avian physiology and poultry farming. By elucidating the impact of stress hormones on egg production and demonstrating the efficacy of gene silencing as a method to enhance reproductive efficiency, this research opens new avenues for improving the productivity and well-being of wild birds in controlled environments. It also underscores the potential of genetic interventions in addressing physiological challenges in wildlife. Presentation: 6/1/2024

Supplementation with ions enhances the efficiency of nucleic acid delivery with cell-penetrating peptides

The successful delivery of therapeutic nucleic acids (NAs) into eukaryotic cells is essential for numerous biomedical applications, including gene therapy, gene silencing, and genome editing. Cell-penetrating peptides (CPPs) have claimed significant attention as delivery vehicles due to their inherent ability to penetrate cellular membranes and efficiently transport cargo, including NAs, into the cells. However, further optimization and a deeper understanding of underlying mechanisms are necessary for such transfection methods. Previous studies have demonstrated that Ca2+ ions can significantly enhance NA delivery efficiency when included in transfection media or CPP/NA nanoparticles during preparation. Similar effects have been observed for Mg2+, but the impact of other ions in this context has not been thoroughly investigated. In this study, we supplemented the CPP/NA formulations with various inorganic biocompatible ions by introducing solutions of the respective salts to colloidal nanoparticles at the preparation stage. Our results indicated that supplementing the CPP/NA formulations with certain salt solutions enhanced the biological effect achieved with NAs while also influencing nanoparticle size, surface charge, complexation stability, and, to some extent, the internalization route. Our findings offer valuable insights for optimizing the formation of CPP nanoparticles to improve NA delivery efficiency.

Runx2 silencing sensitized human renal cell carcinoma cells to ABT-737 apoptosis

The prognostic value of Runt-related transcription factor 2 (Runx2) and its involvement in cell growth and motility have been reported in patients diagnosed with renal cell carcinoma (RCC). Since Runx2 may have the potential to be a target for the purpose of antitumor intervention, there is an urgent need to gain insight into its oncogenic properties. Using human 786-O, Caki-1 and ACHN RCC cells as models, the silencing of cellular Runx2 expression brought about a reduction in cyclin D1 and β-catenin expression, cell growth and migration without any significant cell death. Runx2-silenced cells turned into apoptosis vulnerable in the presence of ABT-737, a BH3 mimetic Bcl-2 inhibitor. Data from biochemical and molecular studies have revealed a positive correlation between Runx2 expression and Akt phosphorylation, Mcl-1 expression, and fibronectin expression. Results of genetic silencing studies have indicated the potential involvement of Mcl-1 and fibronectin in the decision of RCC cell ABT-737 resistance and sensitivity. The regulatory roles of the PI3K/Akt axis in the expression of Mcl-1 and fibronectin were suggested by means of the results taken from experiments involving pharmacological study of the PI3K/Akt. Since overexpression and prognostic roles of Runx2, activated Akt, Mcl-1, fibronectin, cyclin D1, and β-catenin have been revealed in RCC, it is important to explore the precise mechanisms underlying Runx2 oncogenic effects. Although the linking details between Runx2 and PI3K/Akt have yet to be identified, our findings suggest that Mcl-1 and fibronectin are downstream effectors of Runx2 via a regulatory axis of the PI3K/Akt and their promotion of cell growth, migration, and ABT-737 resistance in RCC cells.

The Research Progress of DNA Methylation in the Development and Function of the Porcine Placenta.

The pig is the most widely consumed domestic animal in China, providing over half of the meat supply in food markets. For livestock, a key economic trait is the reproductive performance, which is significantly influenced by placental development. The placenta, a temporary fetal organ, is crucial for establishing maternal-fetal communication and supporting fetal growth throughout pregnancy. DNA methylation is an epigenetic modification that can regulate the gene expression by recruiting proteins involved in gene silencing or preventing transcription factor binding. To enhance our understanding of the molecular mechanisms underlying DNA methylation in porcine placental development, this review summarizes the structure and function of the porcine placenta and the role of DNA methylation in placental development.

Production of active human iduronate-2-sulfatase (IDS) enzyme in Nicotiana benthamiana

Many strategies have been developed to produce high levels of biologically active recombinant proteins in plants for biopharmaceutical purposes. However, the production of an active form of human iduronate-2-sulfatase (hIDS) for the treatment of Hunter syndrome by enzyme replacement therapy (ERT) is challenging due to the requirement for cotranslational modification by a formylglycine-producing enzyme encoded by sulfatase modifying factor 1 (hSUMF1) at the Cys84 residue, which converts it to C(alpha)-formylglycine. In this study, we have shown that hIDS can be highly expressed in N. benthamiana by using different constructs. Among them, BiP-GB1-L-dCBD1-2L-8xHis-L-6xHis-3L-EK-hIDS-HDEL (GB1-CBD1-hIDS) showed a high expression level when transiently co-expressed with the turnip crinkle virus gene silencing suppressor P38 and GB1-fused human calreticulin (GB1-CRT1) as a folding enhancer. The hSUMF1 was co-expressed with hIDS for cotranslational modification. The full-length recombinant proteins were purified using Ni2+-NTA affinity resin followed by enterokinase treatment to obtain tag-free hIDS. The N-terminal fragment was removed using microcrystalline cellulose (MCC) beads. The purified active form of hIDS can successfully cleave the sulfate group from an artificial substrate, 4-nitrocatechol sulfate, at a similar level to commercial hIDS expressed in animal cells. These results suggest that plants could be a promising platform for the production of recombinant hIDS.

A CC-NB-ARC-LRR Gene Regulates Bract Morphology in Cotton.

Bracts are leaf-like structures in flowering plants. They serve multiple functions such as attracting pollinators, aiding tolerance of abiotic stressors, and conducting photosynthesis. While previous studies extensively examine bract function, the molecular mechanisms underlying bract growth remain unknown. Here, the map-based isolation and characterization of a crucial factor responsible for cotton bract development, identified from a mutant known as frego bract (fg), discovered by Frego in 1945 are presented. This gene, named Ghfg, encodes a CC-NB-ARC-LRR (CNL) family protein. Through analysis of bract form in plants with virus-induced gene silencing (VIGS) and transgenic plants, this gene is confirmed to be the causal gene under the fg locus. Furthermore, high-resolution single-cell transcriptomic landscape of cotton bracts is generated, which reveals differences related to auxin in proliferating cells from TM-1 and T582; differences in auxin distribution and ROS accumulation are experimentally verified. These findings suggest that GhFG is in a self-activated state in the fg mutant, and its activity leads to ROS accumulation that impacts auxin distribution and transport. Finally, an island cotton variety with the frego bract trait is developed, demonstrating a novel solution for reducing the high impurity rate caused by bract remnants.

In Vivo HOXB7 Gene Silencing and Cotreatment with Tamoxifen for Luminal A Breast Cancer Therapy.

Acquired resistance and adverse effects are some of the challenges faced by thousands of Luminal A breast cancer patients under tamoxifen (TMX) treatment. Some authors associate the overexpression of HOXB7 with TMX resistance in this molecular subtype, and the knockdown of this gene could be an effective strategy to regain TMX sensitivity. Therefore, we used calcium phosphate hybrid nanoparticles (HNP) for the delivery of short interfering RNA molecule (siRNA) complementary to the HOXB7 gene and evaluated the RNA interference (RNAi) effects associated with TMX treatment in breast cancer in vivo. HNP were prepared by the self-assembly of a methoxy-poly (ethylene glycol)-block-poly (L-glutamic acid) copolymer (PEG-pGlu) and the coprecipitation of CaPO4 to incorporate siRNA. The in vitro cell viability and migration were evaluated prior to in vivo experiments. Further, animals bearing early-stage and advanced Luminal A breast cancer were treated with HNP-siHOXB7, HNP-siHOXB7 + TMX, and TMX. Antitumoral activity and gene expression were evaluated following histopathological, hematological, and biochemical analysis. The HNP were efficient in delivering the siRNA in vitro and in vivo, whilst HOXB7 silencing associated with TMX administration promoted controlled tumor growth, as well as a higher survival rate and reduction in immuno- and hepatotoxicity. Therefore, our findings suggest that HOXB7 can be an interesting molecular target for Luminal A breast cancer, especially associated with hormone therapy, aiming for adverse effect mitigation and higher therapeutic efficacy.

Regulation of maize growth and immunity by ZmSKI3-mediated RNA decay and post-transcriptional gene silencing.

Disease resistance is often associated with compromised plant growth and yield due to defense-growth tradeoffs. However, key components and mechanisms underlying the defense-growth tradeoffs are rarely explored in maize. In this study, we find that ZmSKI3, a putative subunit of the SUPERKILLER (SKI) complex that mediates the 3'-5' degradation of RNA, regulates both plant development and disease resistance in maize. The Zmski3 mutants showed retarded plant growth and constitutively activated defense responses, while the ZmSKI3 overexpression lines are more susceptible to Curvularia lunata and Bipolaris maydis. Consistently, the expression of defense-related genes was generally up-regulated, while expressions of growth-related genes were mostly down-regulated in leaves of the Zmski3-1 mutant compared to that of wild type. In addition, 223 differentially expressed genes that are up-regulated in Zmski3-1 mutant but down-regulated in the ZmSKI3 overexpression line are identified as potential target genes of ZmSKI3. Moreover, small interfering RNAs targeting the transcripts of the defense- and growth-related genes are differentially accumulated, likely to combat the increase of defense-related transcripts but decrease of growth-related transcripts in Zmski3-1 mutant. Taken together, our study indicates that plant growth and immunity could be regulated by both ZmSKI3-mediated RNA decay and post-transcriptional gene silencing in maize.

Elucidating siRNA Cellular Delivery Mechanism Mediated by Quaternized Starch Nanoparticles.

Starch-based nanoparticles are highly utilized in the realm of drug delivery taking advantage of their biocompatibility and biodegradability. Studies have utilized Quaternized starch (Q-starch) for small interfering RNA (siRNA) delivery, in which quaternary amines enable interaction with negatively charged siRNA, resulting in self-assembly complexation. Although reports present numerous applications, the demonstrated efficacy is nonetheless limited due to undiscovered cellular mechanistic delivery. In this study, a deep dive into Q-starch/siRNA complexes' cellular mechanism and kinetics at the cellular level is revealed using single-particle tracking and cell population level using imaging flow cytometry. Uptake studies depict the efficient cellular internalization via endocytosis while a significant fraction of complexes' intracellular fate is lysosome. Utilizing single-particle tracking, it is found that an average of 15% of cellular detected complexes escape the endosome which holds the potential for the integration in the cytoplasmatic gene silencing mechanism. Additional experimental manipulations (overcoming endosomal escape) demonstrate that the complex's disassembly is the rate-limiting step, correlating Q-starch's structure-function properties as siRNA carrier. Structure-function properties accentuating the high affinity of the interaction between Q-starch's quaternary groups and siRNA's phosphate groups that results in low release efficiency. However, low-frequency ultrasound (20kHz) application may have induced siRNA release resulting in faster gene silencing kinetics.

Identification of SLC22A17 DNA methylation hotspot as a potential biomarker in cutaneous melanoma

BackgroundCancer onset and progression are driven by genetic and epigenetic alterations leading to oncogene activation and the silencing of tumor suppressor genes. Among epigenetic mechanisms, DNA methylation (methDNA) is gaining growing interest in cancer. Promoter hypomethylation is associated with oncogene activation while intragenic methDNA can be involved in transcriptional elongation, alternative spicing, and the activation of cryptic start sites. Several genes involved in the modulation of the tumor microenvironment are regulated by methDNA, including the Solute Carrier Family 22 Member 17 (SLC22A17), which is involved in iron trafficking and extracellular matrix remodeling cooperating with the Gelatinase-Associated Lipocalin (NGAL) ligand. However, the exact role of intragenic methDNA in cancer has not been fully investigated. Therefore, the aim of the present study is to explore the role of methDNA in the regulation of SLC22A17 in cutaneous melanoma (CM), used as a tumor model.MethodsCorrelation and differential analyses between SLC22A17 expression and methDNA were performed using the data contained in The Cancer Genome Atlas and Gene Expression Omnibus databases. Functional studies on melanoma cell lines treated with 5-Azacytidine (5-Aza) were conducted to assess the correlation between methDNA and SLC22A17 expression. A validation study on the diagnostic potential of the in silico-identified SLC22A17 methDNA hotspot was finally performed by analyzing tissue samples obtained from CM patients and healthy controls.ResultsThe computational analyses revealed that SLC22A17 was significantly downregulated in CM, and its expression was related to promoter hypomethylation and intragenic hypermethylation. Moreover, SLC22A17 overexpression and hypermethylation of two intragenic methDNA hotspots were associated with a better clinical outcome in CM patients. The correlation between SLC22A17 methDNA and expression was confirmed in 5-Aza-treated cells. In agreement with in silico analyses, the SLC22A17 promoter methylation hotspot showed higher methDNA levels in CM samples compared to nevi. In addition, the methDNA levels of this hotspot were positively correlated with advanced CM.ConclusionsThe SLC22A17 methDNA hotspot could represent a promising biomarker for CM, highlighting the regulatory role of methDNA on SLC22A17 expression. These results pave the way for the identification of novel epigenetic biomarkers and therapeutic targets for the management of CM patients.

A genome-wide association study reveals molecular mechanism underlying powdery mildew resistance in cucumber

BackgroundPowdery mildew is a disease with one of the most substantial impacts on cucumber production globally. The most efficient approach for controlling powdery mildew is the development of genetic resistance; however, few genes associated with inherent variations in cucumber powdery mildew resistance have been identified as of yet.ResultsIn this study, we re-sequence 299 cucumber accessions, which are divided into four geographical groups. A genome-wide association study identifies 50 sites significantly associated with natural variations in powdery mildew resistance. Linkage disequilibrium analysis further divides these 50 sites into 32 linkage disequilibrium blocks containing 41 putative genes. Virus-induced gene silencing and gene expression analysis implicate CsGy5G015960, which encodes a phosphate transporter, as the candidate gene regulating powdery mildew resistance. On the basis of the resequencing data, we generate five CsGy5G015960 haplotypes, identifying Hap.1 as the haplotype most likely associated with powdery mildew resistance. In addition, we determine that a 29-bp InDel in the 3′ untranslated region of CsGy5G015960 is responsible for mRNA stability. Overexpression of CsGy5G015960Hap.1 in the susceptible line enhances powdery mildew resistance and phosphorus accumulation. Further comparative RNA-seq analysis demonstrates that CsGy5G015960Hap.1 may regulate cucumber powdery mildew resistance by maintaining a higher H2O2 level through the depletion of multiple class III peroxidases.ConclusionsHere we identify a candidate powdery mildew-resistant gene in cucumber using GWAS. The identified gene may be a promising target for molecular breeding and genetic engineering in cucumber to enhance powdery mildew resistance.