Diabetic cardiomyopathy (DCM) is a prevalent diabetes-related cardiac complication. miR-671-5p has been shown to mitigate ischemia-reperfusion-induced cardiomyocyte injury. This study investigated the role and underlying mechanisms of miR-671-5p in a DCM cell model established by exposing AC16 cardiomyocytes to high glucose (HG). The miRNA expression dataset GSE210036 from diabetic mouse hearts was analyzed. Cell injury was evaluated by assessing cell viability, apoptosis, and ferroptosis-related alterations. The expression levels and interactions of miR-671-5p, circHUWE1, and CELF1 were examined in the cell model. p38 MAPK activation was further assessed following modulation of the circHUWE1/miR-671-5p/CELF1 axis. Additionally, the m6A modification of circHUWE1 was evaluated. Bioinformatics analysis revealed decreased miR-671-5p expression in diabetic mouse hearts compared to healthy controls. HG treatment downregulated miR-671-5p expression and upregulated the levels of circHUWE1 and CELF1. circHUWE1 upregulation resulted from diminished METTL3-dependent m6A modification. Both miR-671-5p mimic and circHUWE1 knockdown attenuated HG-induced apoptosis and ferroptosis-related alterations. Mechanistically, circHUWE1 elevated CELF1 expression and subsequently activated p38 MAPK by sponging miR-671-5p. The cardioprotective effects of dexmedetomidine (Dex) are associated with the circHUWE1/miR-671-5p/CELF1 axis. In conclusion, the circHUWE1/miR-671-5p/CELF1 axis regulates HG-induced cardiomyocyte apoptosis and ferroptosis-related alterations and represents a novel mechanism underlying Dex-mediated cardioprotection.

Nasopharyngeal carcinoma (NPC) is a malignant tumor with high metastatic potential. Tumor necrosis factor receptor-associated factor 4 (TRAF4) has been identified as an oncogene in various cancers; however, its specific role and molecular mechanism in NPC progression, particularly regarding oxidative stress regulation, remain largely unexplored. In this study, TRAF4 and SETDB1 expression levels were analyzed in NPC cell lines using RT-qPCR and Western blotting. Loss-of-function and gain-of-function assays were performed to evaluate cell proliferation and apoptosis in vitro. The regulatory interaction between TRAF4 and SETDB1 was investigated via co-immunoprecipitation and ubiquitination assays. Mitochondrial function was assessed using MitoSOX, JC-1, and MitoTracker staining to monitor reactive oxygen species (ROS) generation and membrane potential. TRAF4 was significantly upregulated in NPC cell lines compared to immortalized nasopharyngeal epithelial cells. Silencing TRAF4 markedly inhibited NPC cell proliferation and induced apoptosis. Mechanistically, TRAF4 functioned as a critical stabilizer of SETDB1; TRAF4 depletion reduced SETDB1 protein levels by altering its ubiquitination status. Furthermore, SETDB1 knockdown triggered mitochondrial dysfunction, characterized by ROS accumulation, membrane potential collapse, and Cytochrome c release, which could be reversed by the ROS scavenger N-acetyl-L-cysteine (NAC). Importantly, overexpression of SETDB1 rescued the proliferation inhibition and mitochondrial apoptosis induced by TRAF4 silencing. Our findings demonstrate that TRAF4 promotes NPC progression by stabilizing SETDB1 to suppress ROS-dependent mitochondrial apoptosis, highlighting the TRAF4/SETDB1 axis as a promising therapeutic target for NPC treatment.

Steroid-induced osteonecrosis of the femoral head (SONFH) is the most prevalent type of non-traumatic femoral head necrosis. Exosomes (Exo) secreted from stem cells of human exfoliated deciduous teeth (SHED) play crucial roles in biological processes, particularly in tissue regeneration and repair. Previous studies have demonstrated that exosomes from SHED (SHED-Exo) can alleviate dexamethasone (Dex)-induced osteogenic inhibition in bone marrow mesenchymal stem cells (BMSCs). This study aims to further investigate the molecular mechanism by which SHED-Exo rescues BMSCs osteogenic inhibition. Dex-induced BMSCs were used to construct the SONFH model, and SHED-Exo were used for treatment. Differentially expressed miRNAs (DE miRNAs) were identified by miRNA-seq and miRNA target genes were predicted using the database. Regulation was assessed by transfection of miRNA inhibitor and mimics. Alizarin red staining was used to verify osteogenic differentiation capacity, while qPCR and western blotting were employed to analyze osteogenesis-related proteins (OCN, OPN, and RUNX2), EFNA1, and PI3K/AKT pathways. Modulation using PI3K/AKT inhibitors and EFNA1 small interfering RNAs (siRNA) was performed to probe the molecular mechanism of SHED-Exo therapy. miRNA-seq results identified miR-708-5p as a DE miRNA. qPCR confirmed that its expression was down-regulated after Dex treatment and elevated after SHED-Exo treatment. CNTFR was validated as a miR-708-5p target gene through four database predictions and dual luciferase reporter gene results. Transfection of SHED with miR-708-5p inhibitor and mimics demonstrated that miR-708-5p derived from SHED-Exo negatively regulates CNTFR to promote osteogenesis in BMSCs and increases EFNA1 expression, thereby activating the PI3K/AKT pathway. It was further demonstrated that CNTFR is upstream of EFNA1 and negatively regulates the EFNA1/PI3K/AKT pathway by inhibiting PI3K/AKT and interfering with EFNA1 expression. SHED-Exo carries miR-708-5p into BMSCs, promoting EFNA1 expression by negatively regulating CNTFR, which in turn activates the PI3K/AKT pathway and enhances the osteogenic capacity of BMSCs.

Chimeric antigen receptor (CAR) T-cell efficacy requires sustained transgene expression. Cytomegalovirus (CMV) promoters drive strong initial expression but undergo silencing, while elongation factor-1α (EF-1α) promoters resist inactivation. This study evaluated whether transitioning a previously established VHH-based CD19-redirected CAR construct to EF-1α promoter control compromises antitumor functionality. Lentiviral particles encoding identical second-generation CAR constructs (CD19-specific VHH, CD8α transmembrane, 4-1BB costimulatory, CD3ζ signaling domains) under CMV (HCAR-T) or EF-1α (ECAR-T) promoters transduced primary human T cells. Cytotoxicity (effector-to-target ratios 1:1-10:1), antigen-driven proliferation, and cytokine secretion against CD19⁺/CD19⁻ targets were quantified. Promoter substitution from CMV to EF-1α in the matched VHH-based CAR construct did not alter CAR-T cell functionality. ECAR-T and HCAR-T cells exhibited statistically comparable cytotoxicity against CD19⁺ Raji (81.6% ± 2.4 versus 81.8% ± 2.5 at 10:1, respectively) and NALM6 cells (82.2% ± 2.3 versus 80.7% ± 3.5 at 10:1, respectively). Proliferation against Raji (ECAR-T: 94.2% ± 7.54 versus HCAR-T: 91.1% ± 6.03) and NALM6 (ECAR-T: 94.9% ± 4.35 versus HCAR-T: 92.5% ± 6.34) was comparable. IFN-γ, IL-2, and TNF-α secretion against CD19⁺ targets was statistically comparable between ECAR-T and HCAR-T groups and strictly CD19-dependent. EF-1α preserved the antitumor potency of nanobody-based CD19-redirected CAR-T cells, supporting its use as a clinically relevant promoter for further development of this VHH-based CAR-T cell platform.

Macrophages undergo rapid transcriptional reprogramming upon LPS stimulation, but the early regulatory mechanisms (≤6 hours) remain poorly understood. This study investigates the immediate molecular responses in the RAW264.7 murine macrophage cell line, focusing on the interplay between immune activation, cell cycle modulation, and metabolic-epigenetic crosstalk. The metabolic and epigenetic crosstalk mentioned in this study is only inferred from transcriptomic data, and no direct experimental verification was performed. Transcriptomic profiling (RNA-seq) was performed on LPS-stimulated (6-hour) and control macrophages. Differentially expressed genes (DEGs) were analyzed via GO/KEGG enrichment and protein-protein interaction (PPI) networks. Key findings were validated by qPCR and Western blot. Identified 2,715 DEGs (716 upregulated, 1,999 downregulated), with Ikbke identified as a multi-pathway gene (14 pathways). LPS triggered activation of inflammatory pathways (NF-κB, TNF) and downregulation of cell cycle regulators. Ikbke and C5ar1 co-enriched in COVID-19 and viral infection pathways, reflecting their involvement in general innate immune signaling pathways. Transcriptomic findings were validated by qPCR and Western blot, confirming a 6.2-fold induction of Ikbke and significant downregulation of Ezh2. This study identifies Ikbke as a potential correlational candidate of early macrophage responses, linking TLR signaling, metabolic shifts, and viral defense mechanisms. All conclusions in this study are limited to the RAW264.7 immortalized murine macrophage cell line and require further verification in primary cells and in vivo models. These findings in the RAW264.7 cell model provide potential molecular targets for further investigating the modulation of early hyperinflammatory responses.

Oxidized low-density lipoprotein (ox-LDL) and inflammation cause endothelial dysfunction in coronary heart disease (CHD). Although ribosomal protein S5 (RPS5) has been linked to cardiovascular disease, the mechanism by which it functions in the endothelium and its relationship to NF-κB signaling remain unknown. RPS5 was chosen as the hub gene for CHD after differential expression analysis was done on the GSE68506 dataset. The human coronary artery endothelial cells (HCAECs) injury model was established by ox-LDL. In vitro experiments were performed to evaluate the changes in RPS5 expression, apoptosis, inflammatory markers, vascular protective factors, cell proliferation, and Endothelial-to-Mesenchymal Transition (EndMT). Combining the dual luciferase reporter gene assay and the NF-κB nuclear translocation immunofluorescence assay, the effect of RPS5 on the NF-κB pathway was evaluated. Seven ribosomal genes (RPL11, RPL8, RPS13, RPS20, RPS3, RPS5, RPS8) were downregulated in coronary artery disease (CAD) samples in the GSE68506 dataset. In vitro, ox-LDL injury similarly reduced RPS5 expression in HCAECs. Functionally, RPS5 knockdown inhibits cell proliferation and enhances apoptosis and EndMT, while RPS5 overexpression has the opposite effect. Mechanistically, RPS5 overexpression diminished NF-κB nuclear accumulation, lowered κB-WT reporter activity, and reduced the phosphorylation levels of IKKα/β, IκBα, and p65. It also reduced pro-inflammatory mediators and decreased VEGF and HSP70. The pathway dependency was confirmed when LPS-induced NF-κB activation reversed these effects. Through the NF-κB signaling pathway, RPS5 plays a crucial role in controlling inflammation, apoptosis, EndMT, and vascular protection factors in injured HCAECs. Endothelial damage in CHD may be treatable by focusing on the RPS5-NF-κB axis.

Colorectal cancer (CRC) remains a major therapeutic challenge due to toxicity, low tumor specificity, and multidrug resistance. Carvacrol, a natural monoterpenoid phenol with notable anticancer, anti-inflammatory, and antioxidant properties, has emerged as a promising compound. This study evaluated the anticancer efficacy and underlying molecular mechanisms of carvacrol in HT-29 CRC cells and CCD-18Co normal colon fibroblasts. Cell viability was assessed using CVDK-8, migration by wound healing assay, and apoptosis, reactive oxygen species (ROS) levels, and cell cycle distribution by flow cytometry. Expression levels of PDHA1, SOX2, CASP3, POLB, ABCC1, and E2F4 were quantified by qPCR. Bioinformatic analyses were performed using GEPIA2 for overall survival (OS) and disease-free survival (DFS) evaluation and STRING for protein-protein interaction (PPI) network analysis. Carvacrol exhibited dose-dependent cytotoxicity in HT-29 cells (IC50=237.3µM) and significantly inhibited cell migration, whereas its effects on CCD-18Co cells were limited (IC50=425.9µM). In HT-29 cells, carvacrol significantly induced apoptosis, promoted G0/G1 cell cycle arrest, reduced ROS levels, and upregulated CASP3 expression. In contrast, no significant apoptosis, cell cycle arrest, or CASP3 upregulation was observed in CCD-18Co cells, although PDHA1, SOX2, and POLB expression levels were reduced. Bioinformatic analyses revealed that elevated E2F4 expression was associated with poorer OS in CRC and identified a significant interaction sub-network among SOX2, ABCC1, and CASP3. In conclusion, the findings demonstrate that carvacrol effectively inhibits CRC growth while exerting limited effects on normal colon fibroblasts. These results support the further investigation of carvacrol as a potential complementary therapeutic agent for CRC.

Non-small cell lung cancer (NSCLC) represents the predominant form of lung cancer, comprising around 80%-85% of all cases. The Triggering Receptor Expressed on Myeloid Cells 2(TREM2) plays a role in reducing inflammation by influencing the polarization of M2 macrophages. This research aims to explore how TREM2 + macrophages modulate NLRP3 signaling pathway to influence tumor development in NSCLC. In TREM2 knockdown macrophages, the expression levels of NLRP3 at both mRNA and protein levels are significantly increased, which accelerates the cleavage of the downstream signaling molecule Caspase-1 and the release of IL-1β, While diminishing the growth and invasive potential of NSCLC cells. In TREM2-deficient macrophages, treatment with the NLRP3 inflammasome-specific inhibitor MCC950 reverses Caspase-1 cleavage and IL-1β release, thereby restoring the growth and invasive capacity of NSCLC cells. Elevated levels of sTREM2 and IL-1β were detected in the peripheral blood of NSCLC patients. The immunohistochemical staining results also showed that the expression level of TREM2 in the lung cancer tissues of NSCLC patients tended to increase compared to the adjacent normal tissues. Despite numerous studies on TREM2 in neurodegenerative diseases and immune regulation, its specific role within the tumor microenvironment of non-small cell lung cancer (NSCLC) and its relationship with the inflammasome pathway have been infrequently reported. This study elucidates the mechanism by which TREM2 inhibits NLRP3 inflammasome activity in NSCLC, thereby promoting tumor immune evasion and progression. Therefore, TREM2 could serve as a promising target for therapeutic interventions in NSCLC treatment.

Atherosclerosis (AS) is a chronic inflammatory vascular disorder driven by dyslipidemia and characterized by foam cell formation originating from vascular smooth muscle cells (VSMCs). Mitophagy is a critical pathway for the selective clearance of dysfunctional mitochondria and also contributes to macrophage foaming. Tripartite motif-containing protein 65 (TRIM65), a key protein within the TRIM family, plays a significant role in immune and inflammatory responses and is integral to the pathogenesis of AS. In vivo and in vitro models of as were established using ApoE[-/-] mice fed a high-fat diet and mouse aortic vascular smooth muscle (MOVAS) cells treated with oxidized low-density lipoprotein (ox-LDL), respectively. Results indicated a significant increase in TRIM65 expression in atherosclerotic models. The generation of TRIM65[-/-]ApoE[-/-] mice demonstrated that TRIM65 knockdown reduced atherosclerotic plaque burden and lipid accumulation. In vitro experiments revealed that TRIM65 knockdown inhibited the formation of VSMC-derived foam cells while promoting mitochondrial autophagy. Mechanistic investigations showed that TRIM65 facilitates the degradation of key mitochondrial autophagy proteins (e.g., PINK1, Parkin), thereby inhibiting mitochondrial autophagy. This inhibition leads to the accumulation of dysfunctional mitochondria, resulting in excessive ROS generation and exacerbating oxidative stress. By inhibiting mitochondrial autophagy, TRIM65 accelerates the formation of VSMC-derived foam cells, thereby promoting the onset and progression of as. This discovery not only deepens our understanding of the molecular mechanisms of as but also provides theoretical support for positioning TRIM65 as a potential novel therapeutic target for the prevention and treatment of this disease.

OLMALINC has functions in several cancers; however, its function in small cell lung cancer (SCLC) cells and the possible regulatory mechanism are not well understood. Here, we identified the prognostic value of OLMALINC for SCLC and its role in SCLC progression. To identify the expression level of OLMALINC in SCLC, we performed quantitative real-time PCR (RT-PCR) assays in SCLC and adjacent tissue samples. To assess the prognostic potential of OLMALINC, Kaplan-Meier/log-rank tests, Multivariate Cox regression analyses, and time-dependent receiver operating characteristic curves were undertaken. To explore the cell function of OLMALINC in SCLC cells, OLMALINC was inhibited or overexpressed in vitro cell models, followed by apoptosis detection, invasion/migration tests, and determination of neuroendocrine markers (GRP and calcitonin). To identify the possible downstream targets, bioinformatics prediction, analyses of expression level correlation, RIP, RNA pull-down assays, and recovery experiments were conducted. A consistent upregulation of OLMALINC was observed in tumor tissues of SCLC patients and cell lines. OLMALINC upregulation was related to advanced AJCC or VALSG stage, poor overall survival, and poor recurrence-free survival. Gain/loss of function studies of OLMALINC in SCLC cell lines showed OLMALINC decreased cell apoptosis but increased cell migration/invasion and levels of GRP and calcitonin. OLMALINC interacted with SMARCA4, thus activating EZH2. OLMALINC modulated H3K27me3 of CCL2 to regulate CCL2 expression levels. OLMALINC can predict SCLC prognosis and promote SCLC progression.

Gelatin methacryloyl (GelMA), a photosensitive biomaterial, can form three-dimensional (3D) structures with suitable mechanical strength to effectively support cell growth. In this study, the ability of GelMA hydrogels to generate a hypoxic and acidic microenvironment was supported by methods such as visual colorimetry, Western blot, and WST-8 assay. Lung cancer cells (A549) and lung epithelial cells (BEAS-2B) were encapsulated in GelMA hydrogels in a one-step process for 3D culture. CCK-8 assay and Calcein/PI staining results revealed that A549 cells cultured in the 3D system exhibited higher viability than BEAS-2B cells. Moreover, 3D culture promoted stemness acquisition in A549 cells, as evidenced by enhanced drug resistance, increased colony-forming capacity, and upregulated expression of stemness-associated marker genes. We further investigated the regulatory role of the MAPK signaling pathway in this microenvironment. RT-PCR and Western blot analyses confirmed that the GelMA hydrogel-based 3D culture system suppressed the MAPK signaling pathway. These findings suggest that the hypoxic and acidic 3D culture system constructed using GelMA hydrogel promotes both proliferation and stemness maintenance of lung cancer cells, and that inhibition of the MAPK signaling pathway likely contributes critically to this effect.

Knowledge of natural stone promoters in the urine was previously limited, but is essential to develop new targets for better management of calcium oxalate (CaOx) kidney stones. This work, therefore, unveils such information in the urine of CaOx stone formers (patients with stones). Urinary proteins were fractionated by DEAE/GigaQ anion-exchange chromatography, and individual fractions were subjected to multiple crystal assays. The fractions with the summed crystal-promoting score ≥ 3 were subjected to proteomic analysis using nanoLC-ESI-Qq-TOF tandem mass spectrometry (MS/MS). Almost all of the chromatographic fractions (SFQ1-SFQ9) showed promoting effects on CaOx crystallization, growth, aggregation and crystal-cell adhesion. Among them, SFQ2, SFQ8 and SFQ9 provided the greatest summed crystal-promoting score, implicating their roles in stone promotion. MS/MS successfully identified 12, 38 and 6 proteins in fractions SFQ2, SFQ8 and SFQ9, respectively. Among all proteins identified, CD44 antigen, galectin-3-binding protein, kallikrein-1, and protein AMBP were found in more than one fraction, suggesting that they might serve as candidates for the stone promoters. These findings narrow the gap to better understand the pathogenesis and offer opportunities to define new therapeutic targets for better management of CaOx kidney stones.

Long-term peritoneal dialysis (PD) frequently leads to peritoneal fibrosis (PF), resulting in ultrafiltration failure. This study aims to elucidate the molecular mechanisms underlying PD-associated PF and provide new insights for its treatment. Single-cell RNA sequencing (scRNA-seq) data from PD patients in the GEO database were analyzed to examine the cellular composition and gene expression profiles of peritoneal tissues. Human peritoneal mesothelial cells (PMCs) were treated with high glucose (2.5%) to simulate the PD microenvironment. Cellular senescence and related signaling pathways were assessed using SA-β-gal staining, ELISA and Western blot. Regulatory mechanisms were further explored through RNA interference, dual-luciferase reporter assays, and Transwell co-culture experiments. Key findings were validated in a mouse model. scRNA-seq analysis revealed a reduced proportion of PMCs and an increased number of fibroblasts in long-term PD patients, along with activated senescence pathways and elevated RRAS expression in PMCs. In vitro experiments confirmed that high glucose induced PMCs senescence, while RRAS knockdown delayed this process by inhibiting the MEK/ERK pathway. The transcription factor KLF6 was found to directly bind to the RRAS promoter and enhance its transcription. Co-culture experiments further demonstrated that senescent PMCs activated fibroblasts through the paracrine secretion of TGF-β, upregulating the expression of α-SMA, fibronectin, and COL1A1 in fibroblasts. This effect was blocked by KLF6 knockdown or TGF-β receptor inhibition. Animal experiments confirmed that KLF6 knockdown suppressed the RRAS/MEK/ERK pathway, reduced the release of senescence-associated secretory phenotype (SASP) factors, and thereby alleviated peritoneal fibrosis. High-glucose PD fluid induces PMCs senescence by activating the KLF6/RRAS/MEK/ERK axis. Senescent PMCs, promote fibroblast activation via paracrine TGF-β, ultimately driving peritoneal fibrosis. This pathway may serve as a potential therapeutic target for intervening in PF.

Metastasis is the leading cause of death in patients with lung adenocarcinoma (LUAD), highlighting the urgent need to identify novel therapeutic targets. GLIS family zinc finger 1 (GLIS1) is a transcription factor implicated in multiple cancers. However, its role in LUAD remains poorly defined. In this study, we first identified that GLIS1 was significantly upregulated in LUAD tissues. To modulate its expression, lentiviral vectors were constructed to knock down or overexpress GLIS1 in NCI-H1975 and A549 LUAD cell lines, respectively. In vitro gain- and loss-of-function experiments revealed that GLIS1 knockdown significantly inhibited the migratory and invasive capabilities of NCI-H1975 cells. Conversely, GLIS1 overexpression enhanced these malignant phenotypes in A549 cells. In vivo study demonstrated that GLIS1 knockdown substantially reduced liver and lung metastasis of NCI-H1975 cells in mice. Furthermore, we found that downregulation of GLIS1 suppressed LUAD cell migration by inhibiting Homeobox A9 (HOXA9) methylation, which led to the subsequent upregulation of HOXA9 expression. Mechanistically, GLIS1 knockdown transcriptionally suppressed the mRNA level of the DNA methyltransferase DNMT3B, thereby attenuating HOXA9 methylation. Specifically, overexpression of DNMT3B led to DNA hypermethylation and transcriptional suppression of HOXA9, thereby partially rescuing the tumor-suppressive effects resulting from GLIS1 knockdown. Our work is the first to implicate the GLIS1/DNMT3B axis in the epigenetic silencing of HOXA9 in LUAD, highlighting the therapeutic potential of targeting GLIS1.

Prevotella copri, a predominant commensal bacterium in the human gut, exhibits positive or negative correlations with multiple metabolic disorders through its abundance dynamics. Although its association with rheumatoid arthritis pathogenesis is established, the effect of P. copri on bone metabolism remains elusive. This study demonstrates that P. copri exposure induces systemic bone metabolic imbalance in both normal physiological murine models and in vitro-cultured bone marrow-derived adherent cells (BMDCs), which is characterized by exacerbated osteoclastogenesis. Notably, succinate acts as a critical bioactive metabolite, with its specific receptor SUCNR1 being involved in its regulation on bone metabolism. P. copri or succinate intervention triggers bone inflammatory responses, prominently elevating IL-6 expression. Intriguingly, the osteoclastic activation is abolished in Il-6-knockout mice upon P. copri or succinate challenge. These findings delineate a ‌succinate-SUCNR1-IL-6 signaling axis‌ through which P. copri disrupts bone homeostasis, proposing novel therapeutic strategies for bone disorders such as osteoporosis via modulating gut microbiota composition (e.g., diet-mediated Prevotella suppression), reducing succinate bioavailability, and pharmacological antagonism of SUCNR1.