Cellular & Molecular Biology Letters最新文献

Background: Tumor metastasis is a major factor of high recurrence and mortality in clear cell renal cell carcinoma (ccRCC), but its underlying mechanism remains elusive. This study focuses on investigating the impact and underlying molecular mechanisms of MAGUK p55 subfamily member 7 (MPP7) on the metastasis of ccRCC.

Methods: The clinical significance of MPP7 in patients with ccRCC was investigated based on The Cancer Genome Atlas (TCGA), Genotype Tissue Expression Project (GTEx) databases and clinical tissue samples. Slow aggregation, microscopic photography and immunofluorescence (IF) assay were applied to assess the effect of MPP7 on intercellular adhesion, cell morphology, and cytoskeletal F-actin, respectively. Transwell and wound-healing assays were used to detect cell migration and invasion. The quantitative real-time polymerase chain reaction (qRT-PCR), western blot, IF, co-immunoprecipitation (Co-IP), and immunoprecipitation-mass spectrometry (IP-MS) were applied to elucidate the underlying molecular mechanism.

Results: High expression of MPP7 in ccRCC was associated with a better prognosis. Biologically, MPP7 increased intercellular adhesion, affected cell morphology, prevented the overgrowth of F-actin, and significantly inhibited the metastasis of ccRCC cells both in vitro and in vivo. Mechanistically, MPP7 competed with F-actin to bind to α-actinin-4 (ACTN4) through its GuK domain, thereby inhibiting F-actin polymerization. The reduced F-actin aggregation decreased the spatial sequestration of the E3 ligase tripartite motif-containing protein 21 (TRIM21), thus strengthening its access to Snail. The enhanced interaction between TRIM21 and Snail promoted the ubiquitin-proteasome-mediated degradation of Snail, ultimately leading to decreased migration and invasion abilities.

Conclusions: Our work elucidated the role and molecular mechanism of MPP7 in migration and invasion regulation of ccRCC.

Major facilitator superfamily domain-containing protein 2A (MFSD2A) is a central molecular player in maintaining the blood-brain barrier (BBB). It exerts dual protective effects in ischemia-reperfusion injury (IRI): MFSD2A regulates the lipid composition of brain endothelial cell membranes through its sodium-dependent transport of docosahexaenoic acid-conjugated lysophosphatidylcholine (LPC-DHA); this lipid remodeling thereby maintains the characteristic low permeability of the BBB by suppressing caveolae-mediated transcytosis. This review systematically analyzes the tissue distribution patterns of MFSD2A, the protein structural features, and its biological functions both in physiological and pathological conditions. We further reveal its cell type-specific regulatory networks. Notably, acute-phase of IRI induces downregulation of MFSD2A and subsequent BBB leakage. MFSD2A not only serves as a molecular switch to enhance brain-targeted drug delivery (e.g., temporarily inhibiting its activity to improve nanoparticle transport across the BBB) but may also become a therapeutic target for maintaining BBB integrity (e.g., agonist development). This review provides a novel framework for understanding MFSD2A's multidimensional mechanisms in neurological diseases and its potential for clinical translation.

Background: Sperm chromosomes are nonrandomly organized in the cell nucleus, which plays an important role in the regulation of early embryo development, which is determined by the specific localization of sperm chromosomal regions carrying genes with expression crucial at the first contact with ooplasm during fertilization. Thus, the aim of this study is to determine whether the application of selective methods providing high-quality spermatozoa with good motility and/or morphology can increase the frequency of gametes with a specific positioning of chromosomes. For the first time, we used a sequential staining algorithm for consecutive analyses of the same individual spermatozoon with a fixed position, what enables one to achieve full and detailed documentation at the single cell level.

Methods: Semen samples from five normozoospermic males were collected and processed for fractionation via swim up (to select viable and motile spermatozoa) or Percoll density gradient (90%/47%; to select viable sperm with normal motility and morphology). Sperm chromatin protamination was assessed by Aniline Blue (AB) staining, and DNA fragmentation by Acridine Orange (AO) (ssDNA fragmentation) or terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay (ssDNA and dsDNA fragmentation). Then, sequential staining and analyses of the same individual spermatozoon with a fixed position on a slide were performed, in the following order: (i) fluorescence in situ hybridization (FISH) for determination of positioning of chromosomal centromeres: 4, 7, 8, 9, 18, X, and Y, with so-called linear and radial estimations applied, followed by distance measurements between selected pairs of chromosomes; and (ii) immunofluorescent (IF) measurement of global sperm DNA methylation (5mC) and hydroxymethylation (5hmC) levels, which added additional data about the epigenetic layer of the sperm chromosomes' positioning.

Results: Our study demonstrated that high-quality sperm selection methods significantly: (i) increased the frequency of spermatozoa with good chromatin protamination (+ ~25%) and 5mC and 5hmC DNA levels (+ ~9.5%) and (ii) reduced the rate of spermatozoa with ssDNA fragmentation (- ~65%). Motile and morphologically normal spermatozoa showed distinct chromosome repositioning with sex chromosomes shifted to the nuclear periphery, a key chromosomal region of the initial interaction with the ooplasm during fertilization process. Evaluated autosomes revealed various patterns of repositioning.

Conclusions: Our findings underline the validity of methods used for selection of high-quality spermatozoa in assisted reproductive technologies (ART), also in the context of the sperm chromosomal topology and chromatin integrity, crucial at the first steps during fertilization.

Cystic fibrosis (CF) is a life-shortening monogenic disease caused by mutations in the CFTR gene, but the functional expression of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl^-/HCO₃^- channel is determined by more than its genetic sequence. Beyond the well-known folding defect of the common F508del mutation, CFTR activity is dynamically modulated by a network of intracellular signaling pathways that control the channel's gating, trafficking to, and retention at the apical membrane. Foremost is the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) pathway, which drives CFTR opening via phosphorylation of its regulatory (R) domain and coordination by scaffolding proteins (e.g., A-kinase anchoring proteins (AKAPs) and Na⁺/H⁺ exchanger regulatory factor 1 (NHERF1)). Equally important, Ca²⁺-dependent signaling cascades provide complementary fine-tuning: Ca²⁺-bound calmodulin can directly bind and increase the CFTR open probability, Ca²⁺-activated kinases such as Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) and the tyrosine kinase Pyk2 (with Src) can phosphorylate CFTR through noncanonical routes, and signaling intermediates such as IP₃ receptor binding protein released with IP₃(IRBIT) connect Ca²⁺ release to CFTR activation. These cAMP- and Ca²⁺-driven pathways intersect in specialized subcellular nanodomains, enabling precise spatiotemporal regulation of CFTR function. Clinically, although new CFTR modulator drugs have greatly improved outcomes, their effectiveness is limited by mutation-specific responses and incomplete restoration of channel activity. Understanding how cAMP-Ca²⁺ crosstalk governs CFTR in context can reveal novel therapeutic strategies targeting the channel's regulatory microenvironment. This review highlights how compartmentalized cAMP and Ca²⁺ signals orchestrate CFTR function and discusses emerging approaches to harness this insight for better therapies across CF-affected organs.

Background: Microplastics (MPs) have emerged as significant environmental pollutants, posing a threat to ecosystems and humans. The presence of MPs in atherosclerotic plaques, exacerbating cardiovascular risk, has been recently reported. However, the molecular mechanism underlying the effects of MPs on the vascular endothelium are still undefined. In this regard, this study aims to investigate the effects of MPs on endothelial cell function and redox state and the underlying mechanisms.

Methods: Immortalized human aortic endothelial cells (teloHAEC), human umbilical vein endothelial cells (HUVEC), and human coronary artery endothelial cells (HCAEC) were treated with MPs in the form of polyethylene (PE) and polyvinyl chloride (PVC) alone (70 µg/mL) or combined PE (30 µg/mL) + PVC (30 µg/mL) (PE + PVC) for up to 48 h. The effects of MPs on cell viability were evaluated using CCK-8, and its role in endothelial function was evaluated by flow cytometric analyses, enzyme-linked immunosorbent assays (ELISA), and XF HS Seahorse bioanalyzer. Proprotein convertase subtilisin-kexin type 9 (PCSK9) levels were detected by reverse-transcription quantitative polymerase chain reaction (RT-qPCR) and immunoblotting. Molecular involvement of sirtuin 6 (SIRT6) was investigated through gene silencing.

Results: Our study demonstrated that PE and PVC, alone or in combination, upregulated inflammatory mediators monocyte chemoattractant protein-1 (MCP-1), vascular cell adhesion molecule-1 (VCAM1), and intercellular adhesion molecule-1 (ICAM1) (p < 0.001), modulated the expression of autophagy markers anti-autophagy related 5 (ATG5) and p62, impaired mitochondrial metabolism by reducing maximal and basal respiration and adenosine triphosphate (ATP) production (p < 0.001), promoted reactive oxygen species (ROS) accumulation (p < 0.001) and cell cycle perturbations (p < 0.01), and increased apoptosis cell death (p < 0.001). These events were accompanied by a downregulation of sirtuin 6 (SIRT6) expression (p < 0.01) and an upregulation of PCSK9, at protein and messenger RNA (mRNA) levels (p < 0.01). Treatment with the PCSK9 inhibitor (iPCSK9) evolocumab ameliorated MP-induced cellular redox state imbalance, mitochondrial metabolism alteration, and SIRT6 downregulated levels (p < 0.01). SIRT6 transient silencing experiments denied the beneficial effects of iPCSK9 treatment, indicating that the pleiotropic functions of iPCSK9 may occur, at least in part, via modulation of SIRT6 and Forkhead box O3 (FOXO3A) expression levels.

Conclusions: Overall, the results indicate that PCSK9 inhibition via evolocumab exhibits substantial promise in the prevention of MP-induced endothelial dysfunction, suggesting the PCSK9-SIRT6 axis as a new promising pathway to target in preventive strategies for cardiovascular risk caused by plastic pollution.

Background: Hepatocellular carcinoma (HCC) remains a leading cause of cancer-related mortality worldwide, underscoring the urgent demand for novel diagnostic and therapeutic targets. While mitochondrial carriers (MCs) play crucial roles in tumor metabolism, their specific contributions to HCC pathogenesis are poorly understood.

Methods: By leveraging multi-omics analyses, including single-cell sequencing and spatial transcriptomics, SLC25A39 was identified as a key mitochondrial carrier in HCC. To assess its diagnostic potential, receiver operating characteristic (ROC) curves were constructed across multiple retrospective independent cohorts. Functional experiments of HCC cell lines with SLC25A39 knockdown were conducted in vitro (cell proliferation, Transwell migration and invasion, and apoptosis assays) and in vivo (xenograft experiments). For deeper mechanistic insights, we employed proteomic profiling and mitochondrial functional assays. Additionally, the mitochondrial-targeted antioxidant (2-oxo-2-((2,2,6,6-tetramethyl-1-oxyl-piperidin-4-yl)amino)ethyl)triphenylphosphonium chloride (mitoTEMPO) was employed to reverse the observed phenotypes.

Results: SLC25A39 exhibited significant overexpression in HCC tissues, particularly in advanced-stage tumors, and demonstrated robust diagnostic accuracy (area under the curve (AUC) > 0.900 across cohorts). Deficiency of SLC25A39 markedly reduced HCC cell proliferation, migration, and invasion capabilities, triggering caspase-9/3-dependent apoptosis. Consistent with in vitro findings, xenograft models revealed impaired tumor growth upon SLC25A39 suppression. Mechanistically, SLC25A39 deficiency induced mitochondrial dysfunction, characterized by excessive mitochondrial reactive oxygen species (ROS), reduced membrane potential, diminished adenosine triphosphate (ATP) synthesis, aberrant mitochondrial permeability transition pore (mPTP) opening, and cytochrome c release. Notably, mitoTEMPO treatment reversed these effects, restoring mitochondrial redox homeostasis and rescuing malignant phenotypes.

Conclusions: Our study reveals SLC25A39 as a critical regulator of HCC progression via the mitochondrial ROS-cytochrome c-caspase signaling axis, highlighting its potential as a diagnostic biomarker and therapeutic target in HCC.

Background: Aberrant ribosome biogenesis promotes gastric cancer (GC) progression and contributes to chemoresistance by sustaining protein synthesis, upon which GC cell survival depends. However, the regulatory role of cancer-testis-associated long noncoding RNAs (CT-lncRNAs) in modulating ribosome biogenesis in GC remains largely unexplored.

Methods: First, we performed a screening of lncRNAs and identified CT-lncRNA LINC01940 on the basis of integrated expression and survival analyses using The Cancer Genome Atlas (TCGA) data. Subsequently, the impact of LINC01940 on GC progression and chemosensitivity was evaluated using in vitro cell functional assays, patient-derived organoid models, and in vivo subcutaneous tumor xenograft experiments. To further elucidate the underlying mechanisms, we employed a comprehensive approach combining bioinformatics analyses, RNA sequencing, fluorescence in situ hybridization, translation assays, ribosomal DNA (rDNA) transcription assays, methylated RNA immunoprecipitation, co-immunoprecipitation mass spectrometry, fluorescence multiplex immunohistochemistry, and RNA pull-down mass spectrometry.

Results: Normally, testis-specific LINC01940 is aberrantly upregulated in GC and associated with poor prognosis. Functional assays demonstrated that LINC01940 promotes GC cell proliferation and invasion and confers resistance to cisplatin. Mechanistically, LINC01940 is stabilized by methyltransferase 16 (METTL16)/ insulin-like growth factor 2 messenger RNA binding protein 3 (IGF2BP3)-mediated N⁶-methyladenosine (m⁶A) modification, which enhances its ability to act as a scaffold promoting the interaction between the small ubiquitin-like modifier 2 (SUMO2) E3 ligase TATA-box binding protein associated factor 15 (TAF15) and Nucleolar protein 11 (NOL11), promoting the SUMOylation of NOL11 and enhancing its protein stability. This, in turn, increases ribosomal DNA transcription and ribosome biogenesis, thereby promoting GC progression and chemoresistance.

Conclusions: LINC01940 is a cancer-testis lncRNA that promotes GC progression and cisplatin resistance by enhancing ribosome biogenesis via the METTL16/IGF2BP3-TAF15-NOL11 axis. These findings suggest its potential as a prognostic biomarker and therapeutic target in GC.

Background: Obesity, characterized by excessive fat accumulation, represents a global health crisis closely linked to metabolic disorders such as type 2 diabetes, hypertension, and atherosclerosis. tRNA-derived small RNAs (tsRNAs) have recently emerged as important epigenetic regulators, yet their roles in fat deposition remain poorly characterized. This study aims to identify tsRNAs that influence fat accumulation and to elucidate their molecular mechanisms, with a focus on tRF‑Gly‑GCC‑037 (tRF‑Gly) as a candidate regulator of adipocyte differentiation.

Methods: Visceral adipose tissue was collected from obese and lean pigs for comprehensive tRF and tiRNA sequencing. Differential expression analysis identified tRF‑Gly as a highly abundant candidate in obese samples. Functional assays in 3T3‑L1 preadipocytes included both overexpression and knockdown of tRF‑Gly, followed by lipid accumulation measurements and assessment of key adipogenic markers (CEBPα and PPARγ) by quantitative real-time PCR (qRT‑PCR) and western blot. Mechanistically, dual‑luciferase reporter assays, RNA immunoprecipitation (RIP), and nuclear-cytoplasmic protein fractionation were performed to examine how tRF‑Gly modulates the RAC1/JNK2/β‑catenin signaling axis.

Results: tRF‑Gly was significantly upregulated in visceral adipose tissue from obese pigs and ranked among the most abundant tsRNAs. Overexpression of tRF‑Gly in 3T3‑L1 cells and in C57BL/6 mice promoted lipid accumulation and increased CEBPα and PPARγ expression, whereas tRF‑Gly knockdown reduced lipid deposition. Mechanistically, tRF-Gly was suggested to bind RAC1 mRNA with AGO3 involvement, leading to RAC1 silencing. Consistently, RAC1 knockdown phenocopied the adipogenic effects of tRF-Gly, whereas RAC1 overexpression reversed these effects. Furthermore, RAC1 deficiency disrupted the RAC1/JNK2/β‑catenin complex, impaired β‑catenin nuclear translocation, and suppressed Wnt/β‑catenin signaling.

Conclusions: Our findings demonstrate that tRF‑Gly functions as a key regulator of fat accumulation. By silencing RAC1 via AGO3, tRF‑Gly disrupts RAC1/JNK2/β‑catenin complex assembly, prevents β‑catenin nuclear translocation, and downregulates Wnt/β‑catenin signaling, thereby promoting lipid deposition. This study uncovers a novel epigenetic mechanism by which tRF‑Gly controls fat accumulation and suggests that targeting tRF‑Gly may represent a therapeutic strategy for obesity and related metabolic disorders.