Search Results (2,346)

Background/Objectives: Metastatic prostate cancer (PCa) remains a major clinical challenge with limited therapeutic options. The long non-coding RNA H19 has been implicated in regulating cell adhesion molecules and collective migration, key features of metastatic dissemination. This study investigates the role of the Bromodomain and Extra-Terminal (BET) proteins BRD2, BRD3, and BRD4 in the H19-dependent transcriptional regulation of cell adhesion molecules. Currently, the major effects of BET inhibitors require androgen receptor (AR) expression. Methods: H19 was stably silenced in PC-3 (AR-null) and 22Rv1 (AR-positive) castration-resistant PCa cells. The cells were treated with the pan-BET inhibitors JQ1 and OTX015 or the BET degrader dBET6. In vivo, the effects of JQ1 were evaluated in xenograft mouse models. Chromatin immunoprecipitation (ChIP) and RNA-ChIP were used to assess BET protein recruitment and interaction with cell adhesion gene loci and H19. Organotypic slice cultures (OSCs) from fresh PCa surgical specimens were used as ex vivo models to validate transcriptional changes and BRD4 recruitment. Results: BET inhibition significantly reduced the expression of β4 integrin and E-cadherin and cell proliferation in both basal conditions, and following H19 knockdown in PC-3 and 22Rv1 cells. These effects were mirrored in JQ1-treated tumor xenografts, which showed marker downregulation and tumor regression. ChIP assays revealed that BRD4, more than BRD2/3, was enriched on β4 integrin and E-cadherin promoters, especially in regions marked by H3K27ac. H19 silencing markedly enhanced BRD4 promoter occupancy. RNA-ChIP confirmed a specific interaction between BRD4 and H19. These findings were validated in OSCs, reinforcing their clinical relevance. Conclusions: Our study demonstrates that BRD4 epigenetically regulates the H19-mediated transcriptional control of adhesion molecules involved in collective migration and metastatic dissemination. Importantly, these effects are independent of AR status, suggesting that targeting the H19/BRD4 axis may represent a promising therapeutic avenue for advanced PCa. Full article

Resistance to tyrosine kinase inhibitors (TKIs, e.g., sorafenib and lenvatinib) presents a significant hurdle for hepatocellular carcinoma (HCC) treatment, underscoring the need to decipher the underlying mechanisms for improved therapeutic strategies. MicroRNAs (miRNAs) have emerged as critical modulators in HCC progression and TKI resistance. In this study, we report a positive correlation between the expression levels of a tumor suppressor miRNA, miR-142-3p, and increased sensitivity to sorafenib and lenvatinib, supported by clinical data from the BIOSTORM HCC cohort. Overexpression of miR-142-3p in TKI-resistant HCC cells significantly inhibited proliferation and colony formation, induced apoptosis, increased cell cycle arrest at the G2 phase, and reduced migration and invasion by reversing epithelial–mesenchymal transition. Notably, combining miR-142-3p with lenvatinib synergistically inhibited growth in both inherent and acquired TKI-resistant HCC cells by modulating critical signaling pathways, including STAT3, PI3K/AKT, MAPK, YAP1, and by impeding autophagic influx. RNA-sequencing of a TKI-resistant HCC cell line ± miR-142-3p overexpression identified YES1 and TWF1 as direct downstream target genes of miR-142-3p, both of which are key genes associated with drug resistance in HCC. Small interfering RNA (siRNA)-mediated knockdown of these genes mirrored the antitumor effects of miR-142-3p and enhanced TKI sensitivity, with YES1 knockdown decreasing YAP1 phosphorylation, and TWF1 knockdown inhibiting autophagy. Collectively, these findings indicate that restoring miR-142-3p expression or targeting its downstream effectors YES1 and TWF1 offers a promising strategy to overcome drug resistance and improve therapeutic outcome in HCC. Full article

Over the last 40 years, several studies have provided evidence demonstrating that viral vectors can result in effective gene targeting/insertions in a host’s genome. The traditional approaches of gene knock-down, -out, or -in involve an intensive transgenesis process that is plagued by extensive timescales. Plant viruses have the potential to target specific genes and integrate exogenous DNA molecules at the target locus. Their ability to manipulate a host’s genetic material and become a part of it makes them remarkable agents and helpful for molecular and synthetic biology. In this review, we describe how geminivirus-based vectors can be utilized to overcome traditional transgenesis. We highlight the progress that has been made so far and also discuss the hurdles that hinder the employment of geminivirus-based vectors. Furthermore, we conclude with a comparison of geminivirus-based vectors with other plant-derived vectors. Geminivirus-based vectors stand poised to revolutionize plant genome editing by making nucleic acid manipulation cheaper and easier to deploy, thus lessening the major technical constraints, including homology-directed repair (HDR)-mediated genome editing and time-inefficient tissue culture procedures. The insights given in this review illustrate a broader picture of geminiviral vectors, with an emphasis on engineering plant viruses to ease genome editing practices for crop improvements as well as boost experimental timescales from years to months. Full article

Heat stress in dairy cows is aggravated by Global warming, which negatively affects their performance and health, especially high yielding cows are more susceptible to high temperature and humidity in summer. Besides increasing body temperature and reducing feed intake, heat stress also compromises mammary gland function by inducing apoptosis in bovine mammary epithelial cells (BMECs). UFBP1 (Ufm1-binding protein 1) serves as an essential component of ufmylation, is crucial for the preservation of cellular homeostasis. However, little is known about its contribution to heat stress-induced apoptosis in BMECs. Therefore, the present study aimed to elucidate the effect of UFBP1 on heat stress-induced apoptosis through knockdown and overexpression of UFBP1 in BMECs. The results showed that heat stress triggered cell apoptosis (increased apoptosis rate and Bax/Bcl-2 protein expression) and decreased the expression of genes associated with the production of milk fat and protein both in vivo and in vitro studies. Furthermore, UFBP1 silencing aggravated the high-temperature-induced cell damage, and overexpression of UFBP1 attenuated heat stress-induced mitochondrial dysfunction, as evidenced by increased mitochondrial membrane potential (MMP), ATP synthesis and NAD⁺/NADH ratio, as well as the reduced reactive oxygen species (ROS) generation. Importantly, the mitochondrial apoptosis pathway triggered by heat stress was blocked by UFBP1, as indicated by the reduced apoptosis rate and Bax/Bcl-2 protein expression. In addition, UFBP1 restored the expression of milk fat and protein-related genes in heat-stressed BMECs. In conclusion, these findings indicate that UFBP1 may serve as a promising therapeutic target for ameliorating heat stress in dairy cows, thereby providing novel theoretical insights into the mitigation of adverse thermal stress effects on livestock productivity. Full article

Most women with ovarian cancer (OC) develop resistance to platinum chemotherapy, posing a significant challenge to treatment. Matrix metalloproteinase 3 (MMP3) is overexpressed in High-Grade Serous Ovarian Cancer (HGSOC) and is associated with poor survival outcomes; however, its role in platinum resistance remains underexplored. We evaluated the baseline and cisplatin-induced MMP3 transcript and protein levels in cisplatin-resistant OC cells, revealing significantly higher MMP3 levels in cisplatin-resistant cells than in cisplatin-sensitive cells. siRNA-mediated MMP3 knockdown in cisplatin-resistant OC cells significantly reduced viability, proliferation, and invasion, and these effects were further enhanced when combined with cisplatin treatment, indicating a possible synergistic impact on reducing cancer cell aggressiveness; however, chemical MMP3 inhibition did not replicate these effects. RNA sequencing of MMP3-siRNA-treated cisplatin-resistant HGSOC cells revealed 415 differentially expressed genes (DEGs) compared to the negative control, with an additional 440 DEGs identified in MMP3-siRNA HGSOC cells treated in combination with cisplatin. These DEGs were enriched in pathways related to cell cycle regulation, apoptosis, metabolism, stress response, and extracellular matrix organization. Co-immunoprecipitation-coupled mass spectroscopy (IP-MS) identified MMP3-interacting proteins that may contribute to cell survival and chemoresistance in cisplatin-resistant OC. While MMP3-siRNA monotherapy did not reduce tumor growth in vivo, its combination with cisplatin significantly inhibited tumor growth in a cisplatin-resistant HGSOC xenograft model. These findings underscore the multifaceted role of MMP3 in cisplatin resistance, suggesting its involvement in critical cellular processes driving chemoresistance and highlighting the challenges associated with direct MMP3 targeting in therapeutic strategies. Full article

Osteosarcoma (OS) is the most common primary bone malignancy in children and adolescents. Unfortunately, drug resistance limits the efficacy of chemotherapeutic treatment and compromises therapeutic outcomes in a substantial proportion of cases. Aberrant CpG island methylation-associated transcriptional silencing contributes to chemoresistance in pediatric solid tumors. Here, using whole-genome DNA methylation screening on 16 human primary OS specimens, we identify receptor interacting protein kinase-3 (RIPK3), a molecular regulator of the necroptosis programmed cell death pathway, as a gene target of aberrant CpG methylation and demonstrate its role in human OS chemoresistance. We validated these findings via enforced expression and DsiRNA silencing, and evaluated the role of RIPK3 in cisplatin chemosensitivity and necroptosis activation through MLKL phosphorylation. We found that CpG island methylation results in RIPK3 silencing in primary human OS samples and cell lines. Enforced RIPK3 expression significantly enhanced cisplatin cytotoxicity in OS cells and DsiRNA knockdown reversed the cisplatin-sensitive phenotype. In cells with enforced RIPK3 expression, cisplatin treatment significantly increased phosphorylation of both RIPK3 and its target, MLKL, indicative of induction of necroptosis. Here, we identify RIPK3 as an important mediator of chemoresistance in OS and a potential pharmacologic target to improve chemotherapy efficacy in drug-resistant tumors. Full article

Radiotherapy is a cornerstone of colorectal cancer (CRC) treatment; however, its therapeutic efficacy is often compromised by both intrinsic and acquired resistance in CRC cells. This study employed small interfering RNA (siRNA) technology to elucidate the functional roles of BAMBI, GADD34, NFKBIA, and NFKBID in CRC cell lines SW480 and HCT116. We assessed their impact on key cellular processes and radiation sensitivity. Gene silencing of all four target genes significantly suppressed CRC cell proliferation, migration, and invasion. Moreover, siRNA-mediated knockdown enhanced radiation sensitivity, as evidenced by a substantial increase in apoptosis and a marked reduction in cell viability compared with controls. These findings suggest that BAMBI, GADD34, NFKBIA, and NFKBID serve as critical regulators of CRC progression and radiation resistance. Overall, this study provides a mechanistic foundation for further exploration into the pathways underlying radiation resistance and underscores the potential for developing personalized radiotherapy strategies guided by molecular profiling. Full article

Background/Objectives: Cancer cells often display altered energy metabolism. In particular, expression levels and activity of the tricarboxylic acid cycle (TCA cycle) enzymes may change in cancer, and dysregulation of the TCA cycle is a frequent hallmark of cancer cell metabolism. MEMO1, a modulator of cancer metastasis, has been shown to bind iron and regulate iron homeostasis in the cells. MEMO1 knockout changed mitochondrial morphology and iron content in breast cancer cells. Our previous genome-wide analysis of MEMO1 genetic interactions across multiple cancer cell lines revealed that gene sets involved in mitochondrial respiration and the TCA cycle are enriched among the gain-of-function interaction partners of MEMO1. Based on these findings, we measured the TCA cycle metabolite levels in breast cancer cells with varying levels of MEMO1 expression. Methods: ShRNA knockdown assay was performed to test essentiality of key TCA cycle enzymes. TCA metabolites were quantified using liquid chromatography-tandem mass spectrometry (LC-MS/MS) in MDA-MB-231 (high MEMO1), M67-2 (MEMO1 knockdown), and M67-9 (MEMO1 knockout) cells under iron-depleted, basal iron, and iron-supplemented conditions. Results:ACO2 and OGDH knockdowns inhibit cell proliferation, indicating an essential role of the TCA cycle in MDA-MB-231 metabolism. α-Ketoglutarate and citrate levels exhibited an inverse relationship with MEMO1 expression, increasing significantly in MEMO1 knockout cells regardless of iron availability. In contrast, fumarate, malate, and glutamate levels were elevated in MEMO1 knockout cells specifically under low iron conditions, suggesting an iron-dependent effect. Conclusions: Overall, our results indicate that MEMO1 plays a role in regulating the TCA in cancer cells in an iron-dependent manner. Full article

Background: Hepatocellular carcinoma (HCC) is a prevalent and highly lethal form of liver cancer, with limited effective treatment options, particularly in the advanced stages. Immunotherapy using PD-1 inhibitors has emerged as a promising treatment modality, yet a substantial proportion of patients exhibit resistance or fail to respond to such therapies. This study aimed to elucidate the role of G0/G1 Switch 2 (G0S2) in regulating PD-L1 expression in monocytes within the HCC tumor microenvironment and to investigate its impact on the efficacy of PD-1 inhibitors. Methods: Gene expression data among HCC patients treated with PD-1 inhibitors were obtained from the HCC single-cell sequencing database; immunohistochemistry was performed to detect G0S2 expression in liver cancer tissues and adjacent non-tumorous tissues of HCC patients; flow cytometry was utilized to analyze the expression of G0S2, PD-L1, CD206, and CD14 in PBMCs from HCC patients; and CD8⁺T cell proliferation and IFN-γ secretion were used to evaluate the impact of G0S2 knockdown. Results: Utilizing single-cell sequencing data from HCC patients, we identified that G0S2 expression was significantly elevated in the non-responders (NR) compared to responders (R) to PD-1 inhibitor therapy. The immunohistochemical analysis confirmed higher levels of G0S2 in HCC tumor tissues and adjacent non-tumorous tissues, while the flow cytometry revealed the increased expression of G0S2, PD-L1, and CD206 in peripheral blood mononuclear cells (PBMCs) from NR patients compared to R patients and healthy controls. The functional experiments involving the knockdown of G0S2 in the THP-1 monocyte cell line resulted in a significant reduction in PD-L1 expression and a concomitant increase in CD8⁺T cell proliferation and IFN-γ production. Conclusions: These findings indicate that G0S2 facilitates the upregulation of PD-L1 in monocytes, thereby suppressing T cell activity and contributing to resistance against PD-1 inhibitors in HCC. The high expression of G0S2 in peripheral blood monocytes offers a non-invasive and easily detectable biomarker for predicting the efficacy of PD-1 inhibitor therapy. Consequently, targeting G0S2 may enhance the responsiveness to immunotherapy in HCC patients, providing a new avenue for optimizing treatment strategies and improving patient outcomes. Full article

Background: The differentiation process of human mesenchymal stem cells (hMSCs) is regulated by a variety of chemical, physical, and biological factors. These factors activate distinct signaling pathways and transcriptional networks, thereby regulating the lineage-specific differentiation of hMSCs. Objective: This study aims to investigate the role of Immediate Early Response 3 (IER3) in the osteogenic differentiation of human mesenchymal stem cells (hMSCs) and explore the underlying regulatory mechanisms by which IER3 influences osteogenesis. Methods: The expression levels of IER3 and osteogenesis-related genes were quantified when hMSCs were subjected to in vitro osteogenic induction. Then, stable IER3–knockdown hMSCs were generated using IER3–targeted shRNA lentiviral vectors, and the impact of IER3 on osteogenic differentiation was evaluated through both in vitro cell induction and hMSCs subcutaneous implantation model of nude mice. Moreover, RNA–seq and functional inhibition assays were performed to elucidate the signaling pathway through which IER3 regulates the osteogenic differentiation of hMSCs. Results: IER3 expression was significantly downregulated during osteogenic differentiation. Knockdown of IER3 markedly upregulated the expression of ALP and RUNX2, enhancing the osteogenic differentiation capacity of hMSCs, both in vitro and in vivo. Mechanistic studies revealed that IER3 knockdown significantly increased phosphorylated ERK1/2 levels, activating the MAPK/ERK signaling pathway. Furthermore, inhibition of the MAPK/ERK signaling pathway reversed the enhanced osteogenic differentiation observed following IER3 knockdown. Conclusions: Knockdown of IER3 promotes osteogenic differentiation of hMSCs through regulation of the MAPK/ERK signaling pathway, indicating IER3 represents a potential therapeutic target for the treatment of osteoporosis and bone defect-related diseases. Full article

The brown planthopper Nilaparvata lugens, a major rice pest, threatens global food security through rapid reproduction. This study investigates the role of the tramtrack (ttk) gene in male reproductive development and spermatogenesis using RNA interference (RNAi). Gene expression analysis revealed higher ttk levels in testes. RNAi-mediated knockdown of ttk in fourth-instar male nymphs reduced its expression by up to 80%, leading to severely impaired gonad development. Testes, vas deferens, and accessory glands in treated males exhibited 8–89% volume reductions compared to controls, accompanied by a 51–69% decline in sperm count and 60–85% reduction in sperm motility. Consequently, eggs fertilized by treated males showed a 73% decrease in hatching rates, with arrested embryonic development. These findings demonstrate ttk’s critical role in spermatogenesis and gonad maturation in N. lugens, highlighting its potential as an RNAi target for sustainable pest control strategies. Full article

Background: Atherosclerosis is a progressive and complex vascular pathology characterized by cellular heterogeneity, metabolic dysregulation, and chronic inflammation. Despite extensive research, the intricate molecular mechanisms underlying its development and progression remain incompletely understood. Methods: Single-cell RNA sequencing (scRNA-seq) was employed to conduct a comprehensive mapping of immune cell enrichment and interactions within atherosclerotic plaques, aiming to investigate the cellular and molecular complexities of these structures. This approach facilitated a deeper understanding of the heterogeneities present in smooth muscle cells, which were subsequently analyzed using pseudotime trajectory analysis to monitor the developmental trajectories of smooth muscle cell (SMC) subpopulations. An integrative bioinformatics approach, primarily utilizing Weighted Gene Co-expression Network Analysis (WGCNA) and machine learning techniques, identified Cathepsin C (CTSC), transforming growth factor beta-induced protein (TGFBI), and glia maturation factor-γ (GMFG) as critical biomarkers. A diagnostic risk score model was developed and rigorously tested through Receiver Operating Characteristic analysis. To illustrate the functional impact of CTSC on the regulation of plaque formation and SMC viability, both in vitro and in vivo experimental investigations were conducted. Results: An analysis revealed SMCs identified as the most prominent cellular type, exhibiting the highest density of disulfidptosis. Pseudotime trajectory analysis illuminated the dynamic activation pathways in SMCs, highlighting their significant role in plaque development and instability. Further characterization of macrophage subtypes demonstrated intercellular communication with SMCs, which exhibited specific signaling pathways, particularly between the proximal and core areas of plaques. The integrated diagnostic risk score model, which incorporates CTSC, TGFBI, and GMFG, proved to be highly accurate in distinguishing high-risk patients with elevated immune responses and systemic inflammation. Knockdown experiments of CTSC conducted in vitro revealed enhanced SMC survival rates, reduced oxidative stress, and inhibited apoptosis, while in vivo experiments confirmed a decrease in plaque burden and improvement in lipid profiles. Conclusions: This study emphasizes the significance of disulfidptosis in the development of atherosclerosis and identifies CTSC as a potential therapeutic target for stabilizing plaques by inhibiting SMC apoptosis and oxidative damage. Additionally, the risk score model serves as a valuable diagnostic tool for identifying high-risk patients and guiding precision treatment strategies. Full article

Lung adenocarcinoma (LUAD), the predominant subtype of non-small cell lung cancer (NSCLC), presents significant challenges in early diagnosis and personalized treatment. Recent research has focused on the role of the tumor microenvironment, particularly tumor-associated fibroblasts (CAFs), in tumor progression. This study systematically analyzed CAF immune infiltration-related genes to construct a prognostic model for LUAD, confirming its predictive value for patient outcomes. The risk score derived from CAF-related genes (CAFRGs) was negatively correlated with immune microenvironment scores and linked to the expression of immune checkpoint genes, indicating that high-risk patients may exhibit immune escape characteristics. Analysis via the TIDE tool revealed that low-risk patients had more active T-cell immune responses. The risk score also correlated with anti-tumor drug sensitivity, particularly to doramapimod. Notably, COX6A1 emerged as a key gene in the model, with its upregulation associated with immune cell infiltration and immune escape. Further in vitro experiments demonstrated that COX6A1 regulates LUAD cell migration, proliferation, and senescence, suggesting its role in tumor immune evasion. Additionally, further co-culture studies of lung cancer cells and fibroblasts revealed that COX6A1 knockdown promotes the expression of CAF-related cytokines, enhancing CAF infiltration. Overall, this study provides a foundation for personalized treatment of LUAD and highlights COX6A1 as a promising therapeutic target within the tumor immune microenvironment, guiding future clinical research. Full article

Silent Information Regulator 5 (SIRT5) has been established as a crucial regulator of cellular alanylation modification. Furthermore, accumulating evidence suggests that SIRT5 plays a significant regulatory role in key metabolic pathways, including glycolysis, the tricarboxylic acid (TCA) cycle, and fatty acid oxidation, all of which are closely associated with cellular lipid metabolism. Despite these advancements, the specific role of SIRT5 in regulating intramuscular fat (IMF) deposition in goats, as well as the underlying molecular mechanisms, remains largely unexplored. In this study, we cloned the complete coding sequence of the goat SIRT5 gene and, through amino acid sequence alignment, demonstrated its closest phylogenetic relationship with sheep. Additionally, we characterized the higher expression of SIRT5 during the differentiation of goat intramuscular precursor adipocytes. The silencing of SIRT5 by siRNA-mediated knockdown significantly upregulated the expression of lipogenesis-related genes and enhanced lipid deposition in goat intramuscular preadipocytes. Concurrently, SIRT5 deficiency led to the inhibition of cell proliferation and a marked reduction in apoptosis. Interestingly, although overexpression of SIRT5 promoted cell proliferation, it did not significantly alter lipid deposition in goat intramuscular precursor adipocytes. RNA sequencing (RNA-seq) analysis identified a total of 106 differentially expressed genes (DEGs) following SIRT5 silencing in goat preadipocytes, predominantly involved in the Focal adhesion, HIF-1, PI3K-Akt, and MAPK signaling pathways by KEGG pathway enrichment analysis. Notably, we successfully reversed the phenotypic effects observed in SIRT5 knockdown goat precursor adipocytes by inhibiting the PI3K-Akt and MAPK signaling pathways using the AKT inhibitor LY294002 and the p38 MAPK pathway inhibitor PD169316, respectively. In conclusion, our findings demonstrated that SIRT5 may modulate intramuscular fat deposition in goats through PI3k-Akt and MAPK signaling pathways. These results expand the gene regulatory network associated with IMF formation and provide a theoretical foundation for improving meat quality by targeting IMF deposition. Full article

The circadian clock, an intrinsic 24 h cellular timekeeping system, regulates fundamental biological processes, including tumor physiology and metabolism. Cancer stem cells (CSCs), a subpopulation of cancer cells with self-renewal and tumorigenic capacities, are implicated in tumor initiation, recurrence, and metastasis. Despite growing evidence for the circadian clock’s involvement in regulating CSC functions, its precise regulatory mechanisms remain largely unknown. Here, using a human osteosarcoma (OS) model (143B), we have shown that core molecular clock factors are critical for OS stem cell survival and behavior via direct modulation of CSC and lipid metabolic pathways. In single-cell-derived spheroid formation assays, 143B OS cells exhibited robust spheroid-forming capacity under 3D culture conditions. Furthermore, siRNA-mediated depletion of core clock components (i.e., BMAL1, CLOCK, CRY1/2, PER1/2)—essential positive and negative elements of the circadian clock feedback loop—significantly reduced spheroid formation in 143B CSCs isolated from in vivo OS xenografts. In contrast, knockdown of the secondary clock-stabilizing factor genes NR1D1 and NR1D2 had little effect. We also found that knockdown of BMAL1, CLOCK, or CRY1/2 markedly impaired the migration and invasion capacities of 143B CSCs. At the molecular level, silencing of BMAL1, CLOCK, or CRY1/2 distinctly altered the expression of genes associated with stem cell properties and the epithelial–mesenchymal transition (EMT) in 143B CSCs. In addition, disruption of BMAL1, CLOCK, or CRY1/2 expression significantly reduced lipid droplet formation by downregulating the expression of genes involved in lipogenesis (e.g., DGAT1, FASN, ACSL4, PKM2, CHKA, SREBP1), which are closely linked to CSC/EMT processes. Furthermore, transcriptomic analysis of human OS patient samples revealed that compared with other core clock genes, CRY1 was highly expressed in OS tumors relative to controls, and its expression exhibited strong positive correlations with patient prognosis, survival, and LD biogenesis gene expression. These findings highlight the critical role of the molecular circadian clock in regulating CSC properties and metabolism, underscoring the therapeutic potential of targeting the core clock machinery to enhance OS treatment outcomes. Full article