Cellular and Molecular Life Sciences最新文献

Chromatin organization in the mammalian cell nucleus plays a vital role in the regulation of gene expression. The lamina-associated domain at the inner nuclear membrane has been shown to harbor heterochromatin, while the nuclear interior has been shown to contain most of the euchromatin. Here, we show that a sub-set of actively transcribing genes, marked by RNA Pol II pSer2, are associated with Lamin B1 at the inner nuclear envelope in mouse embryonic stem cells (mESCs) and the number of genes proportionally increases upon in vitro differentiation of mESC to olfactory precursor cells. These nuclear periphery-associated actively transcribing genes primarily represent housekeeping genes, and their gene bodies are significantly enriched with guanine and cytosine compared to genes actively transcribed at the nuclear interior. We found the promoters of these gene's to also be significantly enriched with guanine and to be predominantly regulated by zinc finger protein transcription factors. We provide evidence supporting the emerging notion that the Lamin B1 region is not solely transcriptionally silent.

Background: Medulloblastoma (MB), the predominant pediatric malignant brain tumor, remains poorly characterized in terms of its molecular drivers. The lack of reliable early diagnostic biomarkers and effective therapeutic targets continues to pose significant clinical challenges. Recent studies have implicated circular RNAs (circRNAs) as key regulators of diverse oncogenic pathways across multiple solid malignancies. However, the precise processes that underlie circRNAs' function in Sonic Hedgehog (SHH)-driven MB remain unclear.

Methods: RNA-sequencing of three SHH-MB tumors paired with adjacent non-malignant cerebellum was first employed to profile circRNA landscapes, after which hsa_circ_PCNT abundance was precisely quantified by quantitative real-time PCR in an extended cohort of clinical specimens and in established cell lines. The oncogenic and metastatic consequences of altering this transcript were subsequently interrogated through a comprehensive battery of in vitro functional assays and orthotopic xenograft models. To decipher the mechanism, we integrated dual-luciferase reporters, fluorescence in situ hybridization, RNA immunoprecipitation and biotinylated RNA pull-down, thereby demonstrating that hsa_circ_PCNT serves as a cytoplasmic sponge for hsa-miR-133b and consequently alleviates the micRNA-imposed repression of the downstream effector TAGLN2.

Results: Our investigations identified hsa_circ_PCNT as a significantly upregulated circRNA in SHH-MB, with its circular conformation being experimentally validated. Genetic knockdown of this circRNA exerted profound tumor-suppressive effects, including enhanced apoptosis coupled with impaired proliferative capacity, migratory potential, and invasive behavior in vitro. These anti-tumorigenic properties were further corroborated in vivo through xenograft models, where hsa_circ_PCNT depletion markedly attenuated tumor formation. Mechanistically, we established that hsa_circ_PCNT operates as a competitive endogenous RNA by sequestering hsa-miR-133b. Rescue experiments demonstrated phenotypic reversal when hsa-miR-133b levels were experimentally modulated, while subsequent investigations confirmed that TAGLN2 serves as the critical downstream effector mediating these oncogenic processes.

Conclusions: The results show that hsa_circ_PCNT promotes SHH-MB aggression through the hsa-miR-133b /TAGLN2 pathway. In summary, our research demonstrates that hsa_circ_PCNT occupies a crucial position in SHH-MB and emerges as a potential therapeutic target.

Oral mucosal wounds heal faster and with minimal scarring compared to skin injuries, yet the underlying mechanisms remain poorly understood. To explore the cellular and molecular basis of this difference, we performed single-cell RNA sequencing (scRNA-seq) on paired, uninjured human oral mucosa and skin tissues from the same donors. This approach enabled the construction of a comprehensive single-cell transcriptomic atlas, facilitating direct comparison of cellular composition, gene expression profiles, and intercellular communication between the two tissues. Our analysis revealed distinct tissue-specific heterogeneity among keratinocytes, fibroblasts, immune cells, and endothelial cells. Oral keratinocytes exhibited signatures associated with proliferation and metabolic activity, while oral fibroblasts and immune cells expressed gene profiles suggestive of pro-regenerative and anti-fibrotic functions. Cell-cell communication analysis indicated that endothelial cells in oral mucosa participate in interactions that may promote rapid tissue remodeling. Although our data were derived from uninjured tissues, the identified differentially expressed genes and enriched pathways suggest potential regulatory networks that may underlie the distinct wound-healing behaviors of oral mucosa and skin. A subset of these genes was validated by RT-PCR in both autologous and allogeneic samples across different age and sex groups, confirming the robustness and reproducibility of our findings. This study provides the first single-cell transcriptomic comparison of intact human oral mucosa and skin under steady-state conditions, establishing a foundational atlas that reveals intrinsic tissue-specific features and identifies candidate targets for promoting scarless healing.

Skeletal muscle has an innate ability to restore damaged muscle fibers by contributing specific progenitor cells, called muscle satellite cells. Here we show that secretoglobin (SCGB) 3A1, a tumor suppressor gene in various malignancies including rhabdomyosarcoma, is induced just after muscle injury and contributes to damaged muscle fiber regeneration. Lineage tracing of SCGB3A1 in mice show that SCGB3A1-positive cells highly express myosin heavy chain (MyHC)-IIX in damaged fiber area. Scgb3a1-null and Pax7^CreERT2;Scgb3a1^f/f conditional-null mice exhibit defective IIX and IIB fiber regeneration, with a concomitant reduction in the expression of Notch3, a gene important for the maintenance of satellite cell self-renewal pools. Aged Scgb3a1-null mice show reduced size of muscle fibers and mass, resulting in compromised muscle performance as compared to the age-matched wild-type mice. This study reveals that SCGB3A1 is an unexpected novel molecule expressed in muscle satellite cells that contributes to fiber type specific muscle regeneration.

Duchenne muscular dystrophy (DMD) arises from dystrophin deficiency, a crucial component of the dystrophin-glycoprotein complex (DGC) essential for maintaining cellular structural integrity by linking intracellular actin filaments to the basal lamina. Dysfunctions within this complex, coupled with increased inflammatory immune cell infiltration, contribute to the onset of dilated cardiomyopathy (DCM). This cardiac condition, characterized by necrosis and fibrosis, significantly impairs left ventricular function. Despite various treatment approaches, reliable effects on these pathogenic mechanisms remain elusive. RNA-binding proteins play pivotal roles in modulating pathways often dysregulated in cardiac pathology. Notably, HuR, which is upregulated in fibrotic cardiac regions and modulates innate immune system activation, emerges as a promising target. We investigated HuR expression in cardiac tissues of mdx murine model of DMD and assessed the impact of its inhibition with regards to DCM progression. Our findings reveal that HuR is indeed upregulated in mdx mice, and its inhibition leads to attenuation of cardiac fibrosis and improvement in heart function. These preclinical results underscore the potential of targeting HuR for therapeutic intervention to mitigate DCM-associated pathological changes, warranting further exploration for the development of effective treatments.

Objective: Immunosuppressive M2 macrophages in the tumor microenvironment (TME) limit the efficacy of immune checkpoint inhibitors (ICIs), with only ∼20% of lung adenocarcinoma (LUAD) patients benefiting from ICI monotherapy. Targeting macrophage polarization represents a promising strategy to reprogram the TME and enhance antitumor immunity.

Methods: Integrated bioinformatics analysis of TCGA and GEO datasets elucidated the role of IQGAP3 in LUAD progression. A tissue microarray was utilized to compare IQGAP3 expression between tumor and adjacent normal tissues and to evaluate the infiltration relationship between IQGAP3⁺ macrophages and CD8 + T cells. Conditioned medium (CM) from IQGAP3-knockdown macrophage was applied to evaluate its impact on LUAD cell malignancy. Subcutaneous xenograft models tested the impact of IQGAP3 targeting on tumor growth and anti-PD-1 synergy.

Results: IQGAP3 was upregulated in LUAD and correlated with poor survival and reduced ICI benefit. IQGAP3⁺ macrophage infiltration inversely correlated with CD8 + T-cell abundance. CM from macrophage with IQGAP3 knockdown resulted in reduced proliferation, migration and invasion of LUAD cells. In macrophage-cancer cell and macrophage-CD8 + T cell coculture systems, IQGAP3 suppression enhanced macrophage phagocytosis and promoted T cell activation. Mechanistically, IQGAP3 regulates macrophage polarization through binding and sequestering IGF2BP2 at the cell periphery, thereby limiting IGF2BP2-mediated stabilization of FYN mRNA. This reduction in FYN expression led to decreased STAT1 phosphorylation. Targeting IQGAP3 reprogrammed TAMs toward an antitumor phenotype, suppressed tumor growth, and synergized with anti-PD-1 therapy.

Conclusion: IQGAP3 drives immunosuppressive macrophage polarization in LUAD. Its inhibition represents a novel strategy to improve immunotherapy response.

There is growing demand for improved in vitro liver models to better predict in vivo pharmacology, specifically drug disposition mediated by hepatic transporters and assessment of transporter-mediated drug interaction risk. While 2D sandwich-cultured human hepatocytes (SCHH) remain valuable, they are limited to short-term use due to hepatocyte de-differentiation and absence of non-parenchymal cells. Multicellular hepatic spheroids (MHS) offer a promising alternative, but transporter concentrations, functionality, and suitability for hepatobiliary transport studies remain unclear. We evaluated an all-human MHS model, comprised of transporter-certified™ cryopreserved primary human hepatocytes (PHH), Kupffer, stellate, and endothelial cells, for long-term hepatic transporter assessment. Over a 21-day culture period, we monitored transporter concentrations (targeted proteomics), regulation (RNA-seq), localization (immunofluorescence), bile acid profiles (LC-MS/MS), and functional transport (B-CLEAR^®). This is the first report of protein concentrations of 13 transporters in MHS over 21 days directly compared to freshly thawed PHH and SCHH from the same donor. Most transporters declined in MHS compared to PHH, while SCHH maintained or increased transporter concentrations by day 5. However, multidrug resistance-associated protein (MRP) 4 and organic solute transporter (OST)-α/β were upregulated in MHS, likely reflecting adaptation to bile acid accumulation. Bile acid profiling confirmed functional synthesis, metabolism and excretion. Functional MRP2 efflux into sealed canalicular compartments was demonstrated with the MRP2 substrate, 5(6)-carboxy-2',7'-dichlorofluorescein (CDF). Tight junction disruption of canaliculi with Ca²⁺-free buffer resulted in CDF release from canalicular compartments, with partial entrapment within MHS, likely due to the 3D architecture. These findings highlight key strengths and limitations of MHS as a model for assessing hepatobiliary transport.

Propofol is a commonly used anesthetic for sedation during surgery. This drug is reported to exhibit nonanaesthetic immunomodulatory and anti-inflammatory effects. Herein, we investigated the impact of topical propofol delivery with the aim of mitigating psoriatic inflammation. The antipsoriatic potency of propofol was evaluated in a cell-based study in which keratinocytes, macrophages, and neutrophils were used as models. A significant reduction in the proinflammatory effectors interleukin (IL)-6, IL-8, and CXC motif chemokine ligand (CXCL)1 was found in activated keratinocytes (HaCaT) treated with propofol. This reduction could enable baseline control. Immunoblotting suggested that the antioxidant enzymes nuclear factor erythroid 2-related factor (Nrf)2 and heme oxygenase (HO)-1 were involved in the protective effect of propofol on keratinocyte stimulation. The increase in Nrf2 and HO-1 was mediated by kelch-like ECH-associated protein (KEAP)1 downregulation. Propofol presented scavenging activity and decreased 2,2-diphenyl-1-picrylhydrazyl (DPPH) by 47%. The downregulation of cytokines/chemokines in activated macrophages (differentiated THP-1) and mouse neutrophils was also found after propofol treatment. Macrophage migration triggered by the conditioned medium of activated keratinocytes could be blocked with the intervention of propofol. The absorption level of propofol (3 mM) into intact pig skin was 1.2 nmol/mg. Skin deposition was increased to 3.7 nmol/mg after SC lipid removal to mimic psoriasiform skin. In silico molecular docking demonstrated the facile interaction of propofol with ceramides in the stratum corneum (SC). The treatment of imiquimod (IMQ)-sensitized mice with topical propofol suppressed erythema, acanthosis, and macrophage/neutrophil infiltration. Propofol also dramatically decreased cytokine/chemokine levels and epidermal thickness in the lesion. In summary, propofol exhibits anti-inflammatory and antioxidant properties to treat psoriasiform lesions. Topical propofol delivery is useful as an ideal route to accomplish antipsoriatic therapy and avoid systemic effects.

UFMylation, similar to ubiquitination, is a unique post-translational modification which is indispensable in hematopoiesis, neurogenesis and chondrogenesis. However, its role in intervertebral disc development remains unclear. In this study, we focused on DDRGK domain containing protein 1 (DDRGK1), a pivotal component involved in UFMylation, and generated Ddrgk1^fl/fl; Acan-CreER^T2 (Ddrgk1^cKO) mice to explore DDRGK1's regulatory function in the nucleus pulposus and cartilage endplate. We found that Ddrgk1 conditional knockout led to severe retardation of spinal growth, disruption of disc cellularity and initiation of disc degeneration during early postnatal phase. Furthermore, Ddrgk1 conditional knockout in late postnatal phase resulted in profound degeneration of mouse discs, mainly characterized by substantially reduced thickness of cartilage endplate. In addition, Ddrgk1^cKO mice exhibited exacerbated disc degeneration compared to the WT mice after the lumbar spine instability surgery. RNA sequencing of disc cells from Ddrgk1^cKO mice showed upregulation of genes related to apoptosis, matrix metalloproteinase activation, extracellular matrix (ECM) degradation and endoplasmic reticulum (ER) unfolded protein response after Ddrgk1 conditional knockout. Immunohistochemical analysis further verified increased apoptosis, ECM disruption and ER stress in both nucleus pulposus and cartilage endplate after Ddrgk1 conditional knockout. In summary, this study demonstrated that DDRGK1 preserves the normal cellularity and structure of intervertebral discs by regulating the cell fate of nucleus pulposus and cartilage endplate cells, maintaining the ER homeostasis and regulating the metabolic balance of ECM.