Bioinformatics Analysis Research Articles

Fibrinopeptide a promotes the proliferation and migration of vascular smooth muscle cells by regulating the integrin αVβ3/PI3K/AKT signaling pathway.

Atherosclerosis is characterized by subintimal proliferation and migration of vascular smooth muscle cells (VSMCs) in response to stimuli such as coagulation and inflammatory factors. Fibrinopeptide A (FPA), a biomarker for coagulation system activation, is elevated in patients with ischemic heart disease. However, its role in the pathophysiology of cardiovascular disorders remains unclear. This study aimed to investigate the impact of FPA on VSMCs proliferation and migration and elucidate the underlying molecular mechanisms. Transcriptome sequencing and bioinformatics analysis were employed to elucidate molecular pathways. Scratch wound and Transwell assays were performed to evaluate cell migration capacity. Molecular expression patterns were assessed using immunofluorescence, real-time quantitative PCR, and Western blot assays. The differentially expressed genes (DEGs) in the FPA-treated VSMCs were enriched in the cell cycle and PI3K/AKT signaling pathway. FPA treatment enhanced VSMCs' migratory capacity and upregulated integrin αVβ3, PI3K, P-AKT, AKT, Cyclin D1, and PCNA expression. The integrin αVβ3 inhibitor Cyclo-RGDfk effectively suppressed VSMCs migration and reduced the expression levels of these genes in FPA-stimulated VSMCs. These results suggested that FPA promotes the proliferation and migration of VSMCs by regulating the PI3K/AKT signaling pathway through integrin αVβ3.

Targeting the G-quadruplex as a novel strategy for developing antibiotics against hypervirulent drug-resistant Staphylococcus aureus.

The rapid emergence of multiple drug-resistant (MDR) bacterial pathogens and the lack of a novel antibiotic pipeline pose a serious threat to global healthcare. The limited number of established targets further restricts the identification of novel antibiotics to treat life-threatening MDR infections caused by Staphylococcus aureus strains. Therefore, novel targets for developing antibiotics are urgently required. In this study, we hypothesized that the G-quadruplex (G4)-binding ligands can be used as novel antibiotics as their binding can possibly downregulate/block the expression of vital genes. To test this, first we screened the antibiotic properties of representative G4-binding ligands against hypervirulent and MDR S. aureus USA300 and determined the in vitro and in vivo antibacterial activity; and proposed the mechanism of action by applying various microbiological, infection, microscopic, and biophysicochemical techniques. Herein, among screened G4-binding ligands, N-methyl mesoporphyrin IX (NMM) showed the highest antibacterial activity against S. aureus USA300. NMM exhibited a minimum inhibitory concentration (MIC) of 5μM against S. aureus USA300, impacting cell division and the cell wall by repressing the expressions of genes in the division cell wall (dcw) gene cluster. Genome-wide bioinformatics analysis of G4 motifs and their mapping on S. aureus genome, identified the presence of G4-motif in the promoter of mraZ, a conserved master regulator of the dcw cluster regulating the coordinated cell division and cell wall synthesis. Physicochemical assessments using UV-visible, circular dichroism, and nuclear magnetic resonance spectroscopy confirmed that theG4-motif present in the mraZ promoter formed anintramolecular parallel G4 structure, interacting with NMM. In vivo reporter followed by coupled in vitro transcription/translation (IVT) assays confirmed the role of mraZ G4 as a target interacting NMM to impose extreme antibacterial activity against both the gram-positive and -negative bacteria. In-cell and in vivo validation of NMM using RAW264.7 cells and Galleria mellonella; respectively, demonstrated that NMM exhibited superior antibiotic activity compared to well-established antibiotics, with no observed cytotoxicity. In summary, the current study identified NMM as a broad-spectrum potent antibacterial agent and elucidated its plausible mechanism of action primarily by targeting G4-motif in the mraZ promoter of the dcw gene cluster.

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

Abstract 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.

The impact of SEC23A on 5-FU chemotherapy sensitivity and its involvement in endoplasmic reticulum stress-induced apoptosis in colorectal cancer.

Colorectal cancer (CRC) represents a significant global health burden, with chemotherapy resistance representing a significant challenge to effective treatment. SEC23A, a core component of the COPII vesicle trafficking system, is of critical importance with regard to protein transport and cellular homeostasis. Nevertheless, its function in CRC progression and chemoresistance remains uncertain. The present study investigates the correlation between SEC23A expression and sensitivity to 5-fluorouracil (5-FU), a widely used chemotherapeutic agent, with particular emphasis on ER stress-induced apoptosis. A bioinformatic analysis was conducted to evaluate SEC23A expression in CRC and its association with patient prognosis. Chemotherapy sensitivity was predicted using GDSC data and validated experimentally using CRC cell lines with manipulated SEC23A expression. In order to explore the role of SEC23A in acquired drug resistance, patient-derived xenograft (PDX) models and 5-FU-resistant cell lines were employed. Apoptosis assays, cell cycle analysis, and ER stress modulation experiments were performed to elucidate the underlying mechanisms. SEC23A expression was significantly reduced in CRC samples compared to normal tissues. This reduction was linked to a poorer prognosis, including both overall and disease-specific survival. A correlation was observed between low SEC23A expression and increased resistance to 5-FU, as evidenced by both bioinformatic predictions and in vitro experiments. In PDX models, metastatic lesions exhibited decreased SEC23A expression following 5-FU treatment in comparison to primary tumors. Overexpression of SEC23A in 5-FU-resistant cell lines restored sensitivity to the drug and increased apoptosis. Bioinformatic and experimental analyses revealed a robust correlation between SEC23A and ER stress-related apoptotic pathways. Elevated expression of SEC23A was observed to facilitate the accumulation of misfolded proteins in response to 5-FU treatment, which in turn resulted in increased ER stress and apoptosis. SEC23A plays a crucial role in modulating the sensitivity of CRC cells to 5-FU by regulating ER stress-induced apoptosis. Its downregulation contributes to chemoresistance, indicating that SEC23A may serve as a prognostic marker and therapeutic target in CRC. Strategies aimed at upregulating SEC23A or enhancing ER stress may provide new avenues for overcoming chemoresistance and improving treatment outcomes for CRC patients.

Lawsonia intracellularis T3SS effector LI0758, an Rce1 ortholog, activates MAPK and NF-κB signaling in mammalian cells.

Lawsonia intracellularis, a Gram-negative obligate intracellular bacterium causing porcine proliferative enteropathy, possesses a type III secretion system (T3SS), yet only a handful of its substrates have been experimentally characterized. In this study, we identify that LI0758 can be secreted by the Yersinia T3SS, which suppresses yeast growth and activates mitogen-activated protein kinase (MAPK) and nuclear factor-kappa B (NF-κB) signaling pathway in mammalian cells. Bioinformatics analyses indicate that LI0758 is an ortholog of Rce1, a eukaryotic CAAX protein endoprotease, sharing a similar subcellular localization on the endoplasmic reticulum (ER). While displaying unique activity in the yeast a-factor reporter system, LI0758 restores Ras2 localization in Rce1Δ mutant strains, implying functional similarity. Our findings underscore LI0758's pivotal role in activating MAPK pathways and suggest its potential to modulate the localization and function of host CAAX proteins. Further investigation holds promise for elucidating novel bacteria-host interaction mechanisms and fostering the development of innovative therapies against proliferative enteritis.

Efficient Identification of Monoclonal Antibodies Against Rift Valley Fever Virus Using High-Throughput Single Lymphocyte Transcriptomics of Immunized Mice

Background: Rift Valley fever virus (RVFV) is a zoonotic virus that poses a significant threat to both livestock and human health and has caused outbreaks in endemic regions. In humans, most patients experience a febrile illness; however, in some patients, RVF disease may result in hemorrhagic fever, retinitis, or encephalitis. While several veterinary vaccines are being utilized in endemic countries, currently, there are no licensed RVF vaccines or therapeutics for human use. Neutralizing antibodies specifically targeting vulnerable pathogen epitopes are promising candidates for prophylactic and therapeutic interventions. In the case of RVFV, the surface glycoproteins Gc and Gn, which harbor neutralizing epitopes, represent the primary targets for vaccine and neutralizing antibody development. Methods: We report the implementation of advanced 10x Genomics technology, enabling high-throughput single-cell analysis for the identification of rare and potent antibodies against RVFV. Following the immunization of mice with live attenuated rMP-12-GFP virus and successive Gc/Gn boosts, memory B cell populations (both general and antigen-specific) were sorted from splenocytes by flow cytometry. Deep sequencing of the antibody repertoire at a single-cell resolution, together with bioinformatic analyses, was applied for BCR pair selection based on their abundance and specificity. Results: Twenty-three recombinant monoclonal antibodies (mAbs) were selected and expressed, and their antigen-binding capacities were characterized. About half of them demonstrated specific binding to their cognate antigen with relatively high binding affinities. Conclusions: These antibodies could be used for the future development of efficacious therapeutics, as well as for studying virus-neutralizing mechanisms. The current study, in which the single-cell sequencing approach was implemented for the development of antibodies targeting the RVFV surface proteins Gc and Gn, demonstrates the effective applicability of this technique for antibody discovery purposes.

EDF1 accelerates ganglioside GD3 accumulation to boost CD52-mediated CD8+ T cell dysfunction in neuroblastoma

Abstract Background Heterogeneous clinical features and prognosis in neuroblastoma (NB) children are frequently dominated by immune elements. Dysfunction and apoptosis in immune cells result from the exposure to continuous tumor-related antigen stimulation and coinhibitory signals. To date, key factors pointing to the restriction of NB-specific CD8+ T cells remain elusive. Methods We performed bulk-RNA sequencing and lipidomic analyses of children with mediastinal NB. Bioinformatics analysis and biological validation were applied to uncover the underlying mechanism. Results Three subtypes were identified using nonnegative matrix factorization (NMF), among which we highlighted an apoptotic status of infiltrated CD8+ T cells, along with the highest CD52 and EDF1 expression in Cluster3 (C3) subtypes. It was verified that high EDF1 expression in NB cells led to Lactosylceramide (LacCer) accumulation, as well as downstream ganglioside-GD3, which subsequently increased the expression of CD52 and immune checkpoint genes, chemotaxis, and apoptosis-related events in activated CD8+T cells. Mechanistically, EDF1 was recruited as a coactivator to form the NF-κB/RelA/EDF1 complex, which further prevented the promoter region methylation of ST8SIA1, to elevate its transcription. Conclusion These findings characterize abundant GD3 in NB cells, which regulated by the EDF1/RelA/ST8SIA1 axis, is responsible for CD8+ T cell dysfunction. Inhibition of EDF1 may reduce suppressive factors and prevent immune escape of NB cells. Modulating NB-associated GD3 levels through metabolic intervention is beneficial for tuning the depth and duration of responses to current NB therapies. The integration of transcriptomic and lipidomic data offers a more comprehensive understanding of the interaction between LacCer metabolites and the immune status in NB. Graphical Abstract

Integrative bioinformatic approach reveals novel melatonin-related biomarkers for Alzheimer's disease.

Melatonin (MLT) can improve mitophagy, thereby ameliorating cognitive deficits in Alzheimer's disease (AD) patients. Hence, our research focused on the potential value of MLT-related genes (MRGs) in AD through bioinformatic analysis. First, the key cells in the single-cell dataset GSE138852 were screened out based on the proportion of annotated cells and Fisher's test between the AD and control groups. The differentially expressed genes (DEGs) in the key cell and GSE5281 datasets were identified, and the MRGs in GSE5281 were selected via weighted gene coexpression network analysis. After intersecting two sets of DEGs and MRGs, we performed Mendelian randomization analysis to identify the MRGs causally related to AD. Biomarkers were further ascertained through receiver operating characteristic curve (ROC) and expression analysis in GSE5281 and GSE48350. Furthermore, gene set enrichment analysis, immune infiltration analysis and correlation analysis with metabolic pathways were conducted, as well as construction of a regulator network and molecular docking. According to the Fisher test, oligodendrocytes were regarded as key cells due to their excellent abundance in the GSE138852 dataset, in which there were 281 DEGs between the AD and control groups. After overlapping with 3,490 DEGs and 550 MRGs in GSE5281, four genes were found to be causally related to AD, namely, G protein-coupled receptor, family C, group 5, member B (GPRC5B), Methyltransferase-like protein 7A (METTL7A), NF-κB inhibitor alpha (NFKBIA) and RAS association domain family 4(RASSF4). Moreover, GPRC5B, NFKBIA and RASSF4 were deemed biomarkers, except for METTL7A, because of their indistinctive expression between the AD and control groups. Biomarkers might be involved in oxidative phosphorylation, adipogenesis and heme metabolism. Moreover, T helper type 17 cells, natural killer cells and CD56dim natural killer cells were significantly correlated with biomarkers. Transcription factors (GATA2, POU2F2, NFKB1, etc.) can regulate the expression of biomarkers. Finally, we discovered that all biomarkers could bind to MLT with a strong binding energy. Our study identified three novel biomarkers related to MLT for AD, namely, GPRC5B, NFKBIA and RASSF4, providing a novel approach for the investigation and treatment of AD patients.

Anticancer Activity of Enantiomeric Neplanocins A: Exploring the Role of Chirality in Tumor Suppression

Neplanocin A (NPA) is a natural carbocyclic analogue of adenosine that was isolated from Ampullariella regularis, which is known for its antibacterial, antiviral, and anticancer activity. Although the activity of this compound has been demonstrated in many biological models, the mechanism of its anticancer activity is not fully understood. In the current work, we present the comparison of the biological activity of two enantiomers of neplanocin A in the series of cancerous and non-cancerous cell types. In all tested cell lines, the compound with natural stereochemistry, (-)-NPA, was found to be more cytotoxic than its synthetic (+)-NPA derivative; however, sensitivity to neplanocins A varied between cell types. To determine possible reasons for the observed differences in individual cancer cell types, the expression level and effects of individual genes of adenosine-interacting enzymes were analyzed. Bioinformatic analysis of the interaction between (-)-NPA and (+)-NPA with major adenosine-interacting enzymes, such as adenosine kinase (ADK), adenosine deaminases (ADA and ADA2), and S-adenosylhomocysteine hydrolase (SAHH, AHCY), was performed. The molecular docking results revealed differences in the binding energy of the individual enantiomers of neplanocin A with the targets, which sheds new light on the mechanism of action of these adenosine analogues.

Innovative logic “AND” gate gene circuits for bladder cancer treatment: preclinical study

In the evolving field of precision oncology, the synthesis of gene circuits that specifically target cancer cells while preserving normal tissue marks a significant breakthrough. However, traditional approaches typically concentrate on single-gene targets, lacking the directed recognition and control among the intricate networks of signaling pathways. Our study presents a synthetic gene circuit, the Logic “AND” Gate Dual-Target Genetic Circuit (LAG-DTGC), which integrates multiple signals to achieve comprehensive reprogramming of various signaling pathways in bladder cancer (BC) cells. This circuit’s development hinged on detailed bioinformatics analysis, pinpointing more unique biomarkers with similar expression pattern in BC. LAG-DTGC is engineered to selectively activate in cells where these biomarkers are abnormally expressed. Its precision and the remodeling cell behavior capability are further enhanced by incorporating a logic “AND” gate, triggering the circuit only in the presence of these aberrant cancer-specific biomarkers. LAG-DTGC exhibits an extraordinary ability to reprogram cancer cell signaling pathways, turning the cells’ own mechanisms against them for therapeutic effect. This work highlights the potential of synthetic biology in developing precise, less toxic treatments for BC. The LAG-DTGC represents a promising new paradigm in cancer therapy.

Acid sphingomyelinase modulates anxiety-like behavior likely through toll-like receptor signaling pathway.

Recent studies have shown that abnormal activity of acid sphingomyelinase (Asm) has been associated with a range of psychiatric disorders including schizophrenia and depression. However, the role of Asm in the regulation of anxiety remains unclear. In the present study, we employed Asm-knockout (Asm KO) mice to investigate the association between Asm and anxiety using behavioral tests, RNA sequencing, q-PCR, immunohistochemical staining, and other methods. The behavioral results showed that Asm KO mice exhibit enhanced anxiety-like behaviors, such as restricted activity, reduced cumulative times in the central area, diminished exploratory interest, delayed latency to feed, through behavioral tests including open field, novelty-suppressed feeding test, elevated plus maze test, ect. Transcriptional profiling combined with bioinformatics analysis revealed the upregulation of Toll-like receptor signaling pathway related gene including Tlr1/2, Ccl3, Ccl4, Ccl5 and Cd86 in Asm KO mice, which was further confirmed by the detection of activated microglia and astrocytes through iba-1 and GFAP immunohistochemical staining. Collectively, our findings uncover a role for Asm in regulating anxiety-like behavior and suggest that it may be essential for the maintenance of emotional stability, indicating its potential as a promising target for treating anxiety disorders.

An orphan gene is essential for efficient sperm entry into eggs in Drosophila melanogaster.

While spermatogenesis has been extensively characterized in the Drosophila melanogaster model system, very little is known about the genes required for fly sperm entry into eggs. We identified a lineage-specific gene, which we named katherine johnson (kj), that is required for efficient fertilization. Males that do not express kj produce and transfer sperm that are stored normally in females, but sperm from these males enter eggs with severely reduced efficiency. Using a tagged transgenic rescue construct, we observed that the KJ protein localizes around the edge of the nucleus at various stages of spermatogenesis but is undetectable in mature sperm. These data suggest that kj exerts an effect on sperm development, the loss of which results in reduced fertilization ability. Interestingly, KJ protein lacks detectable sequence similarity to any other known protein, suggesting that kj could be a lineage-specific orphan gene. While previous bioinformatic analyses indicated that kj was restricted to the melanogaster group of Drosophila, we identified putative orthologs with conserved synteny, male-biased expression, and predicted protein features across the genus, as well as likely instances of gene loss in some lineages. Thus, kj was likely present in the Drosophila common ancestor. It is unclear whether its role in fertility had already evolved at that time or developed later in the lineage leading to D. melanogaster. Our results demonstrate a new aspect of male reproduction that has been shaped by a lineage-specific gene and provide a molecular foothold for further investigating the mechanism of sperm entry into eggs in Drosophila.

COMMD3 Regulates Copper Metabolism via the ATOX1-ATP7A-LOX Axis to Promote Multiple Myeloma Progression

Background: Multiple myeloma (MM) is a hematologic malignancy characterized by the clonal proliferation of plasma cells, with extramedullary myeloma (EMM) being an aggressive form involving malignant infiltration beyond the bone marrow. Copper metabolism is essential for tumor proliferation and metastasis, with copper metabolism MURR1 domain (COMMD) proteins regulating these processes and maintaining copper homeostasis. Dysregulated copper homeostasis contributes to cancer progression, including MM, with elevated copper levels linked to disease aggressiveness and poor prognosis. This study investigates the role of the COMMD3 in mediating MM cell progression, particularly its influence on copper metabolism. Methods: Comprehensive bioinformatics analyses were conducted on bone marrow and extramedullary samples to determine the expression of COMMD3, which was validated through in vitro and in vivo functional assays. The MM cell lines RPMI8226 and MM1S underwent lentiviral transfection for COMMD3 overexpression and knockdown. RNA sequencing was conducted on COMMD3 knockdown cells to identify differentially expressed genes. Functional assays measured cell proliferation, migration, apoptosis, and copper metabolism, with a non-obese diabetic severe combined immune-deficiency gamma (NSG) mouse xenograft model providing in vivo validation. Results: Elevated COMMD3 expression was correlated with extramedullary myeloma and poor prognosis in MM patients. COMMD3 promoted MM cell proliferation and migration, modulating intracellular copper levels, likely through the ATOX1-ATP7A-LOX copper-metabolism-related pathway. High ATOX1 expression was correlated with worse outcomes, and ATOX1 inhibition abolished COMMD3’s effects. Conclusions: This study highlights the pivotal role of COMMD3 in MM progression, particularly via the ATOX1-ATP7A-LOX axis. These findings provide insights into EMM mechanisms and position COMMD3 as a potential therapeutic target. Future research is needed to validate these findings in larger clinical cohorts and to unravel the precise molecular interactions between COMMD3 and copper metabolism proteins.

Integration of transcriptomics and metabolomics data revealed role of insulin resistant SNW1 gene in the pathophysiology of gestational diabetes.

Gestational Diabetes Mellitus (GDM) is an emerging maternal health problem with increasing incidences. The lack of complete understanding of its pathophysiological mechanisms and novel regulatory biomarkers makes early diagnosis difficult. High-throughput RNA sequencing and computational bioinformatics analyses were conducted to identify novel hub genes, and their regulatory mechanisms were validated through qRT-PCR, western blot, and siRNA-mediated knockdown studies. Intermediate metabolites and circulatory levels of amino acids in the serum of GDM patients and healthy controls were measured. Transcriptomic studies identified SNW1 as the most sensitive and specific biomarker, significantly up-regulated in GDM (fold change = 1.09; p < 0.001). Metabolomic studies indicated significantly elevated gluconeogenesis in GDM, evidenced by decreased levels of alanine and increased levels of pyruvate and glucose compared to controls. siRNA-mediated knockdown of SNW1 in PANC1 cells resulted in significant down-regulation of alanine aminotransferase (ALT/GPT) and insulin receptor substrate (IRS1), while glucose transporters (GLUT2/GLUT4) and insulin (INS) were significantly up-regulated at both mRNA and protein levels. This study identified SNW1 as a novel insulin-resistant gene that induces hyperglycemia by elevating gluconeogenesis and decreasing glucose uptake. SNW1 may be considered a potential therapeutic target with clinical utility for the management of GDM.

Target miRNA identification for the LPL gene in large yellow croaker (Larimichthys crocea).

MicroRNA (miRNA), a conservatively evolved single-stranded non-coding RNA, exerts pivotal control over the appearance of target genes and several biological processes. This study conducted a comprehensive screening of candidate microRNAs (miRNAs) associated with Lipoprotein Lipase (LPL) in the large yellow croaker (Larimichthys crocea), utilizing sophisticated bioinformatics techniques across the species' muscular and hepatic tissues. The bioinformatics analysis facilitated the compilation and examination of miRNA datasets specific to these tissues. The investigation culminated in the identification of miR-84a and miR-1231-5p as key miRNAs that modulate fat hydrolysis, highlighting their potential roles in lipid metabolism. Subsequent in-depth analysis further implicated these miRNAs, along with miR-891a, as prospective targets of LPL, suggesting their integral involvement in the regulation of this critical enzyme. Validation of these bioinformatics predictions was conducted through the construction of double luciferase reporters concealing the LPL 3' untranslated region (3'UTR), substantiating that miR-84a and miR-1231-5p can modulate LPL expression via the LPL 3'UTR. Conversely, miR-891a was not concerned with this regulatory mechanism. Site-directed mutagenesis experiments elucidated the specificity of the interaction sequences. Quantitative PCR assays suggested that miR-84a and miR-1231-5p might influence LPL expression during the starvation phase, intimating the regulatory role of miRNA in fatty acid metabolism within hepatic and muscular tissue under starvation. These findings offer a nuanced understanding of LPL's molecular functionality under stress conditions in fish, emphasizing the regulatory dynamics of miRNA during metabolic stress.

Integrative bioinformatics and experimental analyses identify U2SURP as a novel lactylation-related prognostic signature in esophageal carcinoma.

The lactylation modification has been implicated in several cancer types; however, the role of lactylation modification-related genes in esophageal carcinoma (EC) remains underexplored. Utilizing a set of 16 lactylation modification-related genes, cohorts of patients with EC were stratified into two distinct clusters, characterized by significant disparities in both survival outcomes and the immune microenvironment. An extensive bioinformatics analysis unveiled 382 differentially expressed genes (DEGs) between these two clusters. A subsequent univariate Cox regression analysis identified 24 DEGs specifically associated with lactylation, forming the basis of a constructed lactylation-related score. The resultant lactylation-related score exhibited notable predictive efficacy for survival and other clinicopathological traits, which was validated through calibration curves, Kaplan-Meier survival curves and the Wilcoxon test. Moreover, the lactylation-related score displayed a close correlation with immune cell infiltration in EC. Notable differential expressions of immune checkpoints and regulators were observed between groups stratified by low and high lactylation scores, with the latter exhibiting a more favorable response to anti-PD-1/PD-L1 therapy. Furthermore, the expression profile of U2 snRNP associated SURP domain containing (U2SURP), a constituent of the lactylation-related score, underwent both ex vivo and in vitro validation. The expression of U2SURP was significantly associated with lactylation levels, histological grade and tumor stage. Notably, knockdown of U2SURP expression inhibited the lactylation levels, immune genes IL-1A and IL-1B, proliferation, migration and invasion of EC cells. In conclusion, the lactylation-related score developed in the present study showed promise in predicting the prognosis and immunotherapeutic responses among patients with EC. Moreover, the identification of U2SUPR as a novel oncogene in EC suggests its potential as a prospective therapeutic target for EC treatment.

An integrative analysis of ASCL1 in breast cancer and inhibition of ASCL1 increases paclitaxel sensitivity by activating ferroptosis via the CREB1/GPX4 axis

ObjectiveOur previous study found that Achaete-scute complex homolog 1 (ASCL1) is involved in classifying BC subtypes with different prognostic and pathological characteristics. However, the biological role of ASCL1 in BC still remains largely unexplored. This study aims to elucidate the function of ASCL1 in BC using bioinformatics analyses, as well as in vitro and in vivo experimental approaches.MethodsData from the TCGA, GEO, and Human Protein Atlas databases were utilized to evaluate ASCL1 expression in BC and its association with patient prognosis. Genetic alterations in ASCL1 were assessed through the COSMIC and cBioPortal databases, while the TIMER2.0 database provided insights into the relationship between ASCL1 expression and key gene mutations in BC. The GDSC database was used to examine correlations between ASCL1 levels and sensitivity to standard chemotherapeutic agents. Associations between ASCL1 expression and cytokines, immunomodulatory factors, MHC molecules, and receptors were analyzed using Pearson and Spearman correlation methods. The TIP database was employed to investigate the connection between ASCL1 expression and immunoreactivity scores, and six computational approaches were applied to evaluate immune cell infiltration. Functional assays were conducted on BC cell lines MCF-7 and MDA-MB-231, and nude mouse models were used for in vivo studies.ResultsASCL1 was found to be upregulated in BC and correlated with unfavorable prognosis and mutations in key oncogenes. Its expression was linked to immunomodulatory factors, immune cell infiltration, and immunoreactivity scores in the tumor microenvironment. Additionally, ASCL1 influenced tumor immune dynamics and chemosensitivity in BC. Overexpression of ASCL1 enhanced BC cell proliferation, migration and invasion, while its knockdown had the opposite effect. Notably, inhibition of ASCL1 increased BC cell sensitivity to paclitaxel both in vitro and in vivo. In addition, inhibition of ASCL1 activated ferroptosis in BC, including altered mitochondrial morphology, increased MDA and ROS levels, decreased GSH levels and reduced GSH/GSSG ratio. Mechanistically, inhibition of ASCL1 decreases the phosphorylation of CREB1, thus reducing the expression of GPX4. In summary, inhibition of ASCL1 increases paclitaxel sensitivity by activating ferroptosis via the CREB1/GPX4 axis.ConclusionsASCL1 exerts oncogenic effects in BC and represents a potential therapeutic target for intervention.

ELABELA promotes the migration and homing of bone marrow mesenchymal stem cells to myocardial injury sites through the ERK1/2/miR-299a-5p/Exo70 pathway

BackgroundBone marrow mesenchymal stem cells (BMSCs) hold promise for repairing myocardial injury following acute myocardial infarction (AMI), but their clinical application is hindered by poor migration, homing efficiency, and survival rates. Previously, we demonstrated that ELABELA (ELA), a small peptide, enhances the survival of rat BMSCs under hypoxia-reoxygenation (H/R) conditions by activating ERK1/2. However, the role of ELA in promoting BMSCs migration and homing to injured cardiomyocytes remains unclear.MethodsPrimary BMSCs and neonatal rat ventricular myocytes (NRVMs) were isolated and cultured. NRVMs were exposed to H/R to mimic the microenvironment of AMI in vitro. The migration of BMSCs toward the injured myocardium was assessed in different treatment groups using transwell and chemotaxis assays. Additionally, in vivo studies were performed using a rat myocardial infarction/reperfusion injury (MI/RI) model with DIR-labeled BMSCs. Cardiac repair was evaluated through fluorescence imaging, echocardiography, and histological analysis. Transcriptome sequencing and bioinformatics analysis were employed to identify and validate the mechanisms by which ELA promoted the migration of BMSCs. A dual luciferase assay was used to investigate the interaction between Exo70 and miR-299a-5p. Subsequently, a series of experimental procedures were performed, including sequential silencing of APJ or Exo70, overexpression of miR-299a-5p, inhibition of ERK1/2 phosphorylation, assessment of BMSCs migration through transwell and scratch assays, detection of F-actin polymerization via immunofluorescence, and evaluation of the expression levels of each factor using qPCR and Western blotting.ResultsIn vitro, the migration ability of ELA-pretreated BMSCs was significantly augmented in the H/R environment. ELA pretreatment effectively heightened the homing capacity of BMSCs to the site of myocardial injury and their proficiency in repairing myocardial damage in vivo. Transcriptome sequencing revealed upregulation of Exo70 in ELA pretreated BMSCs, which promoted F-actin polymerization and migration. Overexpression of miR-299a-5p reduced Exo70 expression and impaired BMSCs migration. ELA also activated ERK1/2 phosphorylation, while inhibition of ERK1/2·with U0126 abrogated F-actin polymerization and migration, increasing miR-299a-5p levels and reducing Exo70.ConclusionELA enhances BMSCs migration and homing to injured cardiomyocytes by activating the APJ receptor, promoting ERK1/2 phosphorylation, downregulating miR-299a-5p, and upregulating Exo70, providing a potential therapeutic strategy for improving stem cell-based cardiac repair.

Rutaecarpine alleviates hepatic ischemia‒reperfusion injury in liver transplantation by inhibiting inflammatory response and oxidative stress

BackgroundDonation after circulatory death (DCD) livers are limited by mandatory warm ischemia and are more susceptible to ischemia‒reperfusion injury (IRI). Inflammation and oxidative stress play key roles in the development of hepatic IRI, and Rutaecarpine (Rut) has anti-inflammatory and anti-oxidative stress effects. The aim of this study was to investigate whether Rut can alleviate hepatic IRI in liver transplantation (LT) and to explore the underlying mechanisms.MethodsRat DCD LT and oxygen-glucose deprivation/reoxygenation (OGD/R) cell models were established to clarify the effect of Rut on hepatic IRI. The key molecules involved in the hepatoprotective effects of Rut were identified through joint analysis of data from LT patients and drug targets. The target was further validated by in silico, in vivo and in vitro experiments.ResultsRut significantly alleviated liver dysfunction, pathological injury, and apoptosis and improved the survival rate of the rats subjected to LT. In addition, Rut significantly inhibited inflammatory response and oxidative stress. Rut also had similar effects on OGD/R-induced hepatocyte injury. Mechanistically, bioinformatics analysis and in vivo and in vitro experiments revealed that PDE4B may be a key target by which Rut exerts its protective effect, and molecular docking and cellular thermal shift assay confirmed this result. The function of PDE4B was studied via gene intervention technology, and the results showed that PDE4B can aggravate hepatic IRI. Furthermore, PDE4B overexpression abrogated the protective effect of Rut on the liver in LT.ConclusionRut alleviates hepatic IRI by targeting PDE4B to inhibit inflammation and oxidative stress. These findings highlight the potential of Rut as a drug candidate for the treatment of patients undergoing LT.

Celastrol promotes DNA damage and apoptosis in uterine corpus endometrial carcinoma via promotion of KAT2B-mediated RBPJ acetylation and repression of MCM4 transcription

BackgroundUterine corpus endometrial carcinoma (UCEC) is one of the most frequent female genital malignant tumors. Targeting DNA damage and cell apoptosis are regarded as effective ways for UCEC therapy. Celastrol is a natural anti-cancer product from the Celastraceae plant family, while its role in UCEC has not been investigated.MethodsUCEC cell lines Ishikawa and HEC-1-A were applied and treated with different concentrations of Celastrol. The appropriate and nontoxic concentrations were used for the subsequent experiments. Functional experiments analyzed the cell viability, cell cycle distribution, DNA damage, apoptosis and the expression of related proteins. We determined tumor growth in xenograft nude mice. Bioinformatic analysis, protein coimmunoprecipitation (Co-IP), luciferase assay, cell experiments were performed to reveal the relationship of Celastrol/KAT2B/RBPJ/MCM4 in UCEC.ResultsTreatment of Celastrol inhibited cell viability in a dose-dependent manner, and caused cell cycle arrest, accompanied by the downregulation of CDK2 and cyclin E expression and the upregulation of p21. Celastrol treatment resulted DNA damage and apoptosis in cultured cells, as demonstrated by increased number of TUNEL-positive cells, activity of caspase-3 and expression of cleaved-caspase-9, cleaved PARP1 and γ-H2AX. In xenograft nude mice, Celastrol also repressed tumor growth. Furthermore, lysine acetyltransferase KAT2B was a putative target of Celastrol, and its expression was upregulated by Celastrol in vitro and in vivo. Overexpression of KAT2B in UCEC inhibited cell proliferation and increased DNA damage and apoptosis. KAT2B knockdown overcame the anti-proliferative and pro-apoptotic roles of Celastrol. Moreover, Co-IP demonstrated that KAT2B bound to RBPJ, a transcriptional repressor, and increased the acetylation of RBPJ. RBPJ could bind to the MCM4 promoter to suppress the luciferase activity. Further functional analysis revealed that the functions of KAT2B in UCEC cell proliferation, DNA damage and apoptosis were mediated by MCM4, and Celastrol enhanced RBPJ acetylation and reduced MCM4 expression.ConclusionsThese results underscore that Celastrol is a promising anti-cancer agent in UCEC with preferential anti-proliferative, pro-apoptotic and DNA damage effects through the KAT2B/RBPJ/MCM4 axis, and KAT2B is a promising therapeutic target for UCEC.Graphical abstract