lncRNA PVT1 silencing inhibits gastric cancer cells' progression via enhancing chemosensitivity to paclitaxel.

Gastric cancer (GC) is one of the leading causes of cancer-related deaths worldwide because of its high morbidity and the absence of effective therapies. Even though paclitaxel is a powerful anticancer chemotherapy drug, recent studies have indicated its ineffectiveness against GC cells. Long non-coding RNA (lncRNA) PVT1 has a high expression in GC cells and increases the progression of tumors via inducing drug resistance. In the present study, the effects of the siRNA-mediated lncRNA PVT1 gene silencing along with paclitaxel treatment on the rate of apoptosis, growth, and migration of AGS GC cells were investigated. AGS cells were cultured and then transfected with siRNA PVT1 using electroporation. The MTT test was used to examine the effect of treatments on the viability of cultured cells. Furthermore, the flow cytometry method was used to evaluate the impact of treatments on the cell cycle process and apoptosis induction in GC cells. Finally, the mRNA expression of target genes was assessed using the qRT-PCR method. The results showed that lncRNA PVT1 gene suppression, along with paclitaxel treatment, reduces the viability of cancer cells and significantly increases the apoptosis rate of cancer cells and the number of cells arrested in the G2/M phase compared to the control group. Based on the results of qRT-PCR, combined treatment significantly decreased the expression of MMP3, MMP9, MDR1, MRP1, Bcl-2, k-Ras, and c-Myc genes and increased the expression of the Bax gene compared to the control group. The results of our study showed that lncRNA PVT1 gene targeting, together with paclitaxel treatment, induces apoptosis, inhibits growth, alleviates drug resistance, and reduces the migratory capability of GC cells. Therefore, there is a need for further investigations to evaluate the feasibility and effectiveness of this approach in vivo in animal models.

Silencing ANLN hinders the proliferation, migration, invasion, and angiogenesis of oral squamous cell carcinoma.

BACKGROUND: The actin-binding protein anillin (ANLN) functions as an oncogene in various cancers but has not been fully studied in oral squamous cell carcinoma (OSCC). This study aimed to investigate the expression of ANLN in OSCC tissues and cell lines, to better understand its role in mediating proliferative, angiogenic, invasive, and metastatic capabilities in this type of cancer. METHODS: ANLN mRNA and protein levels were assessed using qPCR and western immunoblotting. The expression intensity of ANLN was evaluated using immunohistochemical (IHC) staining. Biological functional assays were employed to characterize the behavior of OSCC cells influenced by ANLN. Additionally, comprehensive bioinformatics analysis, including GO analysis and KEGG enrichment analysis, was performed on differentially expressed genes in ANLN-mediated pathways. RESULTS: OSCC tumors and cell lines exhibited higher ANLN expression. Silencing of ANLN significantly suppressed OSCC cell proliferation, as evidenced by a significant reduction in the Ki-67 index both in vitro and in vivo. The migration and invasive ability of OSCC cells were markedly diminished, coinciding with a decrease in epithelial-mesenchymal transition activity. ANLN was also found to promote angiogenic activity in OSCC cells, partly through synergistic effects mediated by vascular endothelial growth factor A (VEGFA). Downregulation of ANLN expression led to decreased VEGFA levels, resulting in reduced angiogenesis characterized by fewer vascular branches. CONCLUSIONS: Our findings highlight the promising role of ANLN as a biomarker for both diagnostic and prognostic in OSCC. Targeting ANLN with inhibitory strategies could impede the oncogenesis processes at the core of OSCC development, presenting significant opportunities for advancing therapeutic interventions.

Silencing LINC00663 inhibits inflammation and angiogenesis through downregulation of NR2F1 via EBF1 in bladder cancer.

This study is to elucidate the effect of the LINC00663/EBF1/NR2F1 axis on inflammation and angiogenesis in bladder cancer (BC) and related molecular mechanisms. After transfection, functional experiments were conducted to test cell proliferation and invasion, tube formation ability, and content of inflammatory factors, Snail, E-cadherin, and VEGFA. Meanwhile, the relationships among LINC00663, EBF1, and NR2F1 were predicted and verified. In addition, xenograft experiments in nude mice were performed to observe the oncogenicity of 5637 BC cells in vivo. In BC tissues and cells, LINC00663 and NR2F1 were upregulated. Silencing NR2F1 or LINC00663 repressed cell proliferation and invasion, weakened vascular mimicry in vitro, decreased inflammatory factor, Snail, and VEGFA levels, and increased expression of E-cadherin. LINC00663 positively regulated NR2F1 expression through EBF1. Additionally, in vivo experiments showed that NR2F1 upregulation reversed the suppression effects of LINC00663 silencing on tumour growth, inflammation, and angiogenesis. Silencing LINC00663 decreased NR2F1 expression by mediating EBF1, thereby inhibiting BC inflammation and angiogenesis.

miR-7160 inhibits gastric cancer cell proliferation and metastasis by silencing SIX1.

Upregulation of homeoprotein SIX1 in gastric cancer (GC) is related to tumour proliferation and invasion. MicroRNA-7160 (miR-7160) is a homeoprotein SIX1-targeting miRNA that downregulates miR-7160, leading to cancer development. Total gastric cancer samples were collected from six patients, and relative expression levels of SIX1 mRNA and miRNAs were analysed by qRT-PCR. To evaluate the regulation of SIX1 by miR-7160, pGL3-SIX1-mut, pGL3-SIX1, and miR-7160 mimics transfected into cells using lipofectamine 2000. After transfection, proliferation and apoptosis in cultured cells were assessed using the nuclear TUNEL staining and CCK8 reagent, respectively. We demonstrated that the downregulation of miR-7160 in human gastric cancer cells is related to the upregulation of SIX1 mRNA. In gastric cancer cell lines, miR-7160 overexpression could downregulate the expression and inhibit cancer cell proliferation and growth in vitro. However, overexpression of miR-7160 did not increase gastric cancer cell apoptosis. In vitro downregulation of SIX1 decreased vimentin, N-cadherin, and other EMT-related gene expression and increased E-cadherin expression. In brief, miR-7160, by targeting SIX1, inhibits gastric cancer proliferation and cell growth in vitro, which provides an idea for introducing a new treatment option for gastric cancer.

Heterochromatin: Hiding from the remodeling machines.

In this issue of Molecular Cell, Sahu et al.1 find that shielding heterochromatin from SWI/SNF chromatin remodelers is essential to maintain and epigenetically propagate pre-existing heterochromatin domains, whereas SWI/SNF action protects facultative heterochromatic regions from premature silencing.

Epigenetic silencing of ZIC4 unveils a potential tumor suppressor role in pediatric choroid plexus carcinoma.

Zic family member ZIC4 is a transcription factor that has been shown to be silenced in several cancers. However, understanding the regulation and function of ZIC4 in pediatric choroid plexus tumors (CPTs) remained limited. This study employed data mining and bioinformatics analysis to investigate the DNA methylation status of ZIC4 in CPTs and its correlation with patient survival. Our results unveiled ZIC4 methylation as a segregating factor, dividing CPT cohorts into two clusters, with hyper-methylation linked to adverse prognosis. Hyper-methylation of ZIC4 was confirmed in a choroid plexus carcinoma-derived cell line (CCHE-45) by bisulfite sequencing. Furthermore, our study demonstrated that demethylating agent and a histone methyltransferase inhibitor could reverse ZIC4 silencing. RNA sequencing and proteomic analysis showed that ZIC4 over-expression influenced genes and proteins involved in immune response, antigen processing and presentation, endoplasmic reticulum stress, and metabolism. Functionally, re-expressing ZIC4 negatively impacted cell proliferation and migration. Ultimately, these findings underscore ZIC4 hyper-methylation as a prognostic marker in CPTs and shed light on potential mechanisms underlying its tumor suppressor role in CPC. This insight paves the way for novel therapeutic targets in treating aggressive CPTs.

Transcriptional silencing in Saccharomyces cerevisiae: known unknowns.

Transcriptional silencing in Saccharomyces cerevisiae is a persistent and highly stable form of gene repression. It involves DNA silencers and repressor proteins that bind nucleosomes. The silenced state is influenced by numerous factors including the concentration of repressors, nature of activators, architecture of regulatory elements, modifying enzymes and the dynamics of chromatin.Silencers function to increase the residence time of repressor Sir proteins at silenced domains while clustering of silenced domains enables increased concentrations of repressors and helps facilitate long-range interactions. The presence of an accessible NDR at the regulatory regions of silenced genes, the cycling of chromatin configurations at regulatory sites, the mobility of Sir proteins, and the non-uniform distribution of the Sir proteins across the silenced domain, all result in silenced chromatin that only stably silences weak promoters and enhancers via changes in transcription burst duration and frequency.These data collectively suggest that silencing is probabilistic and the robustness of silencing is achieved through sub-optimization of many different nodes of action such that a stable expression state is generated and maintained even though individual constituents are in constant flux.

Paracoccidioides lutzii Infects Galleria mellonella Employing Formamidase as a Virulence Factor.

The formamidase (FMD) enzyme plays an important role in fungal thriving by releasing a secondary nitrogen source as a product of its activity. In Paracoccidioides species, previous studies have demonstrated the upregulation of this enzyme in a wide range of starvation and infective-like conditions. However, Paracoccidioides lutzii formamidase has not yet been defined as a virulence factor. Here, by employing in vivo infections using an fmd-silenced strain in Galleria mellonella larvae model, we demonstrate the influence of formamidase in P. lutzii's immune stimulation and pathogenicity. The formamidase silencing resulted in improper arrangement of the nodules, poor melanogenesis and decreased fungal burden. Thus, we suggest that formamidase may be a piece composing the process of molecular recognition by Galleria immune cells. Furthermore, formamidase silencing doubled the observed survival rate of the larvae, demonstrating its importance in fungal virulence in vivo. Therefore, our findings indicate that formamidase contributes to Galleria's immune incitement and establishes the role of this enzyme as a P. lutzii virulence factor.

Effects of SvAPETALA1-5 gene on floral organ development in Senecio vulgaris.

Asteraceae is a large class of eudicots with complex capitulum, and little is known regarding the molecular regulation mechanism of flower development. APETALA1(AP1) belongs to the MADS-box gene family and plays a key role in plant floral induction and floral organ development. In this study, the bioinformatics and tissue-specific expression of AP1 homologous gene SvAP1-5 in Senecio vulgaris were analyzed. Based on VIGS technology, SvAP1-5 gene silencing plants were created, and SvAP1-5 was overexpressed in Solanum nigrum. The results of bioinformatics analysis showed that SvAP1-5 gene had typical MADS-box and K-box structure, and contains FUL motif and paleoAP1 motif at the C-terminal. SvAP1-5 belongs to the euFUL branch of AP1 gene. qRT-PCR results showed that SvAP1-5 was expressed in bracts, petals and carpels, and was highly expressed in carpels. Compared with the control group, SvAP1-5 gene silencing resulted in irregular petal dehiscence, increased stigma division, and carpel dysplasia. The fruit development of SvAP1-5 overexpressing S.nigrum plants was abnormal, and the hyperplastic tissue similar to fruit appeared. In summary, SvAP1-5 gene may be involved in the development of petals and carpels and plays an important role during the development of S.vulgaris.

Transcriptome, hormonal, and secondary metabolite changes in leaves of DEFENSE NO DEATH 1 (DND1) silenced potato plants.

DEFENSE NO DEATH 1 (DND1) is a cyclic nucleotide-gated ion channel protein. Earlier, it was shown that the silencing of DND1 in the potato (Solanum tuberosum L.) leads to resistance to late blight, powdery mildew, and gray mold diseases. At the same time, however, it can reduce plant growth and cause leaf necrosis. To obtain knowledge of the molecular events behind the pleiotropic effect of DND1 downregulation in the potato, metabolite and transcriptome analyses were performed on three DND1 silenced lines of the cultivar 'Désirée.' A massive increase in the salicylic acid content of leaves was detected. Concentrations of jasmonic acid and chlorogenic acid and their derivatives were also elevated. Expression of 1866 genes was altered in the same way in all three DND1 silenced lines, including those related to the synthesis of secondary metabolites. The activation of several alleles of leaf rust, late blight, and other disease resistance genes, as well as the induction of pathogenesis-related genes, was detected. WRKY and NAC transcription factor families were upregulated, whereas bHLHs were downregulated, indicating their central role in transcriptome changes. These results suggest that the maintenance of the constitutive defense state leads to the reduced growth of DND1 silenced potato plants.

Molecular approaches for the management of papaya ringspot virus infecting papaya: a comprehensive review.

Papaya ringspot virus (PRSV) is a catastrophic disease that causes huge yield losses in papaya cultivation around the world. Yield losses in severely infected plants can be upto 100%. Because of this disease, papaya cultivation has been shifted to other crops in some areas of the world. Many conventional methods and breeding approaches are used against this disease, which turns out to be less effective. Considering the yield loss caused by PRSV in papaya, it is high time to focus on alternative control methods. To implement effective management strategies, molecular approaches such as Marker Assisted Breeding (MAS) or transgenic methods involving post-transcriptional gene silencing targeting the genome viz., coat protein, replicase gene, or HC Pro can be pursued. However, the public's reluctance to widely accept the transgenic approach due to health and environmental concerns necessitates a consideration of non-transgenic alternatives. Prioritizing safety and ensuring efficient virus control, non-transgenic approaches which encompass cross-protection, genome editing, and topical applications of dsRNA to induce gene silencing within the host, can be adopted. This review aims to provide comprehensive insights of various molecular tools used in managing PRSV which in turn will help in sustainable agriculture.

SIRT1 silencing ameliorates malignancy of non-small cell lung cancer via activating FOXO1.

Non-small cell lung cancer (NSCLC), being the most prevalent and lethal malignancy affecting the lungs, poses a significant threat to human health. This research aims at illustrating the precise role and related mechanisms of silent information regulator type-1 (SIRT1) in NSCLC progression. The expression pattern of SIRT1 in NSCLC cell lines was examined using quantitative real-time polymerase chain reaction and western blotting. Functional assays in NSCLC cell lines validated the biological capabilities of SIRT1 on malignant phenotypes, and its impact on tumorigenicity was further evaluated in vivo. In addition, the FOXO1 inhibitor AS1842856 was applied to verify the role of SIRT1 on FOXO pathway in vitro. SIRT1 expression was prominently elevated in NSCLC cell lines. The depletion of SIRT1 retarded the capabilities of proliferation, migration and invasion, while enhancing apoptosis in NSCLC cells. Furthermore, SIRT1 silencing restricted the tumorigenesis of NSCLC in vivo. Additionally, AS1842856 treatment ameliorated the inhibitory effect of SIRT1 deficiency on malignant phenotypes in NSCLC cells. SIRT1 deletion exerted an anti-oncogenic role in NSCLC via activation of FOXO1.

DNA methylation governs the sensitivity of repeats to restriction by the HUSH-MORC2 corepressor.

The human silencing hub (HUSH) complex binds to transcripts of LINE-1 retrotransposons (L1s) and other genomic repeats, recruiting MORC2 and other effectors to remodel chromatin. How HUSH and MORC2 operate alongside DNA methylation, a central epigenetic regulator of repeat transcription, remains largely unknown. Here we interrogate this relationship in human neural progenitor cells (hNPCs), a somatic model of brain development that tolerates removal of DNA methyltransferase DNMT1. Upon loss of MORC2 or HUSH subunit TASOR in hNPCs, L1s remain silenced by robust promoter methylation. However, genome demethylation and activation of evolutionarily-young L1s attracts MORC2 binding, and simultaneous depletion of DNMT1 and MORC2 causes massive accumulation of L1 transcripts. We identify the same mechanistic hierarchy at pericentromeric α-satellites and clustered protocadherin genes, repetitive elements important for chromosome structure and neurodevelopment respectively. Our data delineate the epigenetic control of repeats in somatic cells, with implications for understanding the vital functions of HUSH-MORC2 in hypomethylated contexts throughout human development.

Silencing of GhSINAT5 Reduces Drought Resistance and Salt Tolerance in Cotton.

The SEVEN IN ABSENTIA (SINA) E3 ubiquitin ligase is widely involved in drought and salt stress in plants. However, the biological function of the SINA proteins in cotton is still unknown. This study aimed to reveal the function of GhSINAT5 through biochemical, genetic and molecular approaches. GhSINAT5 is expressed in several tissues of cotton plants, including roots, stems, leaves and cotyledons, and its expression levels are significantly affected by polyethylene glycol, abscisic acid and sodium chloride. When GhSINAT5 was silenced in cotton plants, drought and salinity stress occurred, and the length, area and volume of the roots significantly decreased. Under drought stress, the levels of proline, superoxide dismutase, peroxidase and catalase in the GhSINAT5-silenced cotton plants were significantly lower than those in the non-silenced control plants, whereas the levels of hydrogen peroxide and malondialdehyde were greater. Moreover, the expression of stress-related genes in silenced plants under drought stress suggested that GhSINAT5 may play a positive role in the plant response to drought and salt stress by regulating these stress response-related genes. These findings not only deepen our understanding of the mechanisms of drought resistance in cotton but also provide potential targets for future improvements in crop stress resistance through genetic engineering.

Inherited defects of piRNA biogenesis cause transposon de-repression, impaired spermatogenesis, and human male infertility.

piRNAs are crucial for transposon silencing, germ cell maturation, and fertility in male mice. Here, we report on the genetic landscape of piRNA dysfunction in humans and present 39 infertile men carrying biallelic variants in 14 different piRNA pathway genes, including PIWIL1, GTSF1, GPAT2, MAEL, TDRD1, and DDX4. In some affected men, the testicular phenotypes differ from those of the respective knockout mice and range from complete germ cell loss to the production of a few morphologically abnormal sperm. A reduced number of pachytene piRNAs was detected in the testicular tissue of variant carriers, demonstrating impaired piRNA biogenesis. Furthermore, LINE1 expression in spermatogonia links impaired piRNA biogenesis to transposon de-silencing and serves to classify variants as functionally relevant. These results establish the disrupted piRNA pathway as a major cause of human spermatogenic failure and provide insights into transposon silencing in human male germ cells.

Systematic Review of Genetic Substrate Reduction Therapy in Lysosomal Storage Diseases: Opportunities, Challenges and Delivery Systems.

BACKGROUND: Genetic substrate reduction therapy (gSRT), which involves the use of nucleic acids to downregulate the genes involved in the biosynthesis of storage substances, has been investigated in the treatment of lysosomal storage diseases (LSDs). OBJECTIVE: To analyze the application of gSRT to the treatment of LSDs, identifying the silencing tools and delivery systems used, and the main challenges for its development and clinical translation, highlighting the contribution of nanotechnology to overcome them. METHODS: A systematic review following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) reporting guidelines was performed. PubMed, Scopus, and Web of Science databases were used for searching terms related to LSDs and gene-silencing strategies and tools. RESULTS: Fabry, Gaucher, and Pompe diseases and mucopolysaccharidoses I and III are the only LSDs for which gSRT has been studied, siRNA and lipid nanoparticles being the silencing strategy and the delivery system most frequently employed, respectively. Only in one recently published study was CRISPR/Cas9 applied to treat Fabry disease. Specific tissue targeting, availability of relevant cell and animal LSD models, and the rare disease condition are the main challenges with gSRT for the treatment of these diseases. Out of the 11 studies identified, only two gSRT studies were evaluated in animal models. CONCLUSIONS: Nucleic acid therapies are expanding the clinical tools and therapies currently available for LSDs. Recent advances in CRISPR/Cas9 technology and the growing impact of nanotechnology are expected to boost the clinical translation of gSRT in the near future, and not only for LSDs.

Effects of Modulating BMP9, BMPR2, and AQP1 on BMP Signaling in Human Pulmonary Microvascular Endothelial Cells.

Pulmonary arterial hypertension (PAH) is a chronic disease characterized by a progressive increase in mean pulmonary arterial pressure. Mutations in the BMPR2 and AQP1 genes have been described in familial PAH. The bone morphogenetic proteins BMP9 and BMP10 bind with high affinity to BMPR2. Administration of BMP9 has been proposed as a potential therapeutic strategy against PAH, although recent conflicting evidence dispute the effect of such a practice. Considering the involvement of the above molecules in PAH onset, progression, and therapeutic value, we examined the effects of modulation of BMP9, BMPR2, and AQP1 on BMP9, BMP10, BMPR2, AQP1, and TGFB1 expression in human pulmonary microvascular endothelial cells in vitro. Our results demonstrated that silencing the BMPR2 gene resulted in increased expression of its two main ligands, namely BMP9 and BMP10. Exogenous administration of BMP9 caused the return of BMP10 to basal levels, while it restored the decreased AQP1 protein levels and the decreased TGFB1 mRNA and protein expression levels caused by BMPR2 silencing. Moreover, AQP1 gene silencing also resulted in increased expression of BMP9 and BMP10. Our results might possibly imply that the effect of exogenously administered BMP9 on molecules participating in the BMP signaling pathway could depend on the expression levels of BMPR2. Taken together, these results may provide insight into the highly complex interactions of the BMP signaling pathway.

Strigolactones Negatively Regulate Tobacco Mosaic Virus Resistance in Nicotiana benthamiana.

Strigolactones (SLs) are plant hormones that regulate diverse developmental processes and environmental responses in plants. It has been discovered that SLs play an important role in regulating plant immune resistance to pathogens but there are currently no reports on their role in the interaction between Nicotiana benthamiana and the tobacco mosaic virus (TMV). In this study, the exogenous application of SLs weakened the resistance of N. benthamiana to TMV, promoting TMV infection, whereas the exogenous application of Tis108, a SL inhibitor, resulted in the opposite effect. Virus-induced gene silencing (VIGS) inhibition of two key SL synthesis enzyme genes, NtCCD7 and NtCCD8, enhanced the resistance of N. benthamiana to TMV. Additionally, we conducted a screening of N. benthamiana related to TMV infection. TMV-infected plants treated with SLs were compared to the control by using RNA-seq. The KEGG enrichment analysis and weighted gene co-expression network analysis (WGCNA) of differentially expressed genes (DEGs) suggested that plant hormone signaling transduction may play a significant role in the SL-TMV-N. benthamiana interactions. This study reveals new functions of SLs in regulating plant immunity and provides a reference for controlling TMV diseases in production.

Use of tRNA gene barriers improves stability of transgene expression in CHO cells.

Instability of transgene expression is a major challenge for the biopharmaceutical industry, which can impact yields and regulatory approval. Some tRNA genes (tDNAs) can resist epigenetic silencing, the principal mechanism of expression instability, and protect adjacent genes against the spread of repressive heterochromatin. We have taken two naturally occurring clusters of human tDNAs and tested their ability to reduce epigenetic silencing of transgenes integrated into the genome of Chinese hamster ovary (CHO) cells. We find sustained improvements in productivity both in adherent CHO-K1 cells and in an industrially relevant CHO-DG44 expression system (Apollo X, FUJIFILM Diosynth Biotechnologies). We conclude that specific tDNA clusters offer potential to mitigate the widespread problem of production instability.

Silencing of KIAA1429, a N6-methyladenine methyltransferase, inhibits the progression of colon adenocarcinoma via blocking the hypoxia-inducible factor 1 signalling pathway.

KIAA1429 is an important 'writer' of the N6-methyladenine (m6A) modification, which is involved in tumour progression. This study was conducted to explore the mechanism of action of KIAA1429 in colon adenocarcinoma (COAD). KIAA1429-silenced COAD cell and xenograft tumour models were constructed, and the function of KIAA1429 was explored through a series of in vivo and in vitro assays. The downstream mechanisms of KIAA1429 were explored using transcriptome sequencing. Dimethyloxalylglycine (DMOG), an activator of HIF-1α, was used for feedback verification. The expression of KIAA1429 in COAD tumour tissues and cells was elevated, and KIAA1429 exhibited differential expression at different stages of the tumour. Silencing of KIAA1429 inhibited the proliferation, migration, and invasion of HT29 and HCT116 cells. The expression levels of NLRP3, GSDMD and Caspase-1 were decreased in KIAA1429-silenced HT29 cells, indicating the pyroptotic activity was inhibited. Additionally, KIAA1429 silencing inhibited the growth of tumour xenograft. Transcriptome sequencing and reverse transcription quantitative polymerase chain reaction revealed that after KIAA1429 silencing, the expression of AKR1C1, AKR1C2, AKR1C3 and RDH8 was elevated, and the expression of VIRMA, GINS1, VBP1 and ARF3 was decreased. In HT29 cells, KIAA1429 silencing blocked the HIF-1 signalling pathway, accompanied by the decrease in AKT1 and HIF-1α protein levels. The activation of HIF-1 signalling pathway, mediated by DMOG, reversed the antitumour role of KIAA1429 silencing. KIAA1429 silencing inhibits COAD development by blocking the HIF-1 signalling pathway.