Intervertebral disc degeneration (IVDD) is closely associated with cellular senescence and defective autophagic degradation, but the molecular heterogeneity and functional significance of lysosome-related alterations remain incompletely understood. We integrated bulk transcriptomic datasets (GSE56081 and GSE70362) and single-cell RNA-sequencing data (GSE153066) using consensus clustering, weighted gene coexpression network analysis, machine-learning algorithms, immune-signature analysis, and covariate-adjusted correlation testing. Candidate genes were evaluated in human and rat disc tissues, and PLD3 was further examined by knockdown and overexpression in H2O2-treated nucleus pulposus cells (NPCs). Lysosomal function was assessed using LysoTracker staining, cathepsin activity, and autophagy-related markers, and the preclinical effects of lithocholic acid (LA) were evaluated in cultured NPCs and a rat needle-puncture model. Two lysosome-related molecular subtypes were identified: a senescence/inflammation-enriched subtype and a metabolism-enriched subtype. The lysosomal gene-signature score was positively associated with the senescence score after adjustment for total cellular transcript counts (partial r = 0.422; empirical permutation p = 0.0005), supporting coordinated transcriptional activation rather than enhanced degradative function. HYAL1, MMD, PLD3, and ANK3 were prioritized as candidate hub genes. PLD3 knockdown aggravated H2O2-induced lysosomal impairment, matrix degeneration, and senescence, whereas PLD3 overexpression produced opposing protective effects. LA partially improved acidic lysosomal compartments, cathepsin activity, autophagic degradation, and senescence-associated changes in vitro and attenuated degeneration-associated histological and molecular alterations in vivo. These findings reveal distinct lysosome-related phenotypes in IVDD, functionally support PLD3 as a contributor to lysosomal homeostasis, and suggest that lysosome-modulating interventions may have therapeutic potential.
BACKGROUND:Metabolic pathways are crucial in hepatocellular carcinoma (HCC) pathogenesis, but causal metabolic genes remain unclear. This study used Summary data-based Mendelian Randomization (SMR) and colocalization to identify metabolism-related genetic loci influencing HCC risk. METHODS:Differentially expressed genes in hepatic malignancy phenotype versus normal tissues from TCGA and GTEx were analyzed. Metabolism-related candidates were examined via SMR and colocalization using multi-omics data: methylation (mQTL), expression (eQTL), and protein (pQTL) quantitative trait loci. RESULTS:Multi-omics integration identified NIT2 as a key metabolic regulator for HCC. The cg13016775 locus of NIT2 was associated with elevated HCC risk at gene (OR = 1.618, 95% CI: 1.199-2.182) and protein (OR = 4.432, 95% CI: 1.783-11.018) levels. Colocalization supported a shared causal variant (PPH4 > 0.6), linking NIT2 to hepatocarcinogenesis via metabolic regulation. CONCLUSIONS:This study provides multi-omics evidence for NIT2 as a potential causal gene in HCC, enhancing understanding of metabolic contributions to HCC pathogenesis and highlighting integrative genomics for uncovering causal relationships.
OBJECTIVE:This study aims to elucidate the mechanism through which prostate cancer-associated transcript 6 (PCAT6) modulates immune escape in triple-negative breast cancer (TNBC), focusing on its interaction with IGF2BP1 and PD-L1. METHODS:We analyzed 68 paired TNBC clinical specimens and utilized TNBC cells (BT-549, MDA-MB-468) with lentivirus-mediated gene manipulation. Functional assays included cell counting kit-8, EdU incorporation, Transwell migration/invasion, and co-culture with activated CD8+ T cells. Immune function was assessed through LDH release, ELISA (IFN-γ and granzyme B), and flow cytometry. Molecular interactions were investigated via RNA immunoprecipitation, RNA pulldown, fluorescence in situ hybridization, and actinomycin D-based mRNA stability assays. RESULTS:PCAT6 was markedly upregulated in TNBC tissues and cell lines, correlating with advanced tumor stage and lymph node metastasis. PCAT6 knockdown restrained tumor cell proliferation, migration, and invasion while downregulating PD-L1 expression. In co-culture systems, PCAT6 depletion enhanced CD8+ T cell cytotoxicity, evidenced by increased inflammatory factor secretion and elevated IFN-γ+ CD8+ T cell proportion. Mechanistically, PCAT6 interacted with IGF2BP1 in the cytoplasm and promoted IGF2BP1-mediated stabilization of PD-L1 mRNA. Rescue experiments confirmed that PCAT6 required IGF2BP1 to sustain PD-L1 expression and mRNA stability. CONCLUSION:PCAT6 facilitates TNBC immune escape by enhancing PD-L1 mRNA stability through IGF2BP1, identifying the PCAT6/IGF2BP1/PD-L1 axis as a potential therapeutic target for breast cancer immunotherapy.
BACKGROUND:Adenovirus vectors (AdVs) are widely used and have an advantage of large insert capacity compared with adeno-associated virus vectors. However, AdVs have scarcely been used in genome-editing knock-in strategies because of low efficiency. METHODS:Novel AdVs possessing a very large, 3.7 kb donor DNA fragment and six or eight multiplex gRNA expression units were developed for CRISPR/Cas9-mediated knock-in to correct a phenylalanine hydroxylase (Pah) gene in a Pahenu2 phenylketonuria mouse model. These AdVs were co-infected to Hepa1-6 cells or liver cells in vivo together with an AdV expressing either native Cas9 or Cas9 nickase (Cas9n) for double-nicking cleavage. RESULTS:In vitro knock-in of the AdVs carrying 3.7 kb donor DNA and six gRNA units targeting the cell genome was observed in both cases using Cas9 and Cas9n, though their efficiencies were low. Therefore, we generated AdVs carrying an additional two gRNA units that cleave the donor DNA terminus in the AdV genome via native Cas9 or Cas9 nickase. The knock-in efficiency increased approximately twofold for both vectors and reached a maximum of 8% for native Cas9 without selection. Newborn phenylketonuria model mice were intravenously administered the knock-in AdV together with the native-Cas9 AdV. Although the knock-in efficiency by homologous recombination occurred in only approximately 1% of hepatocytes, blood phenylalanine levels were reduced by up to 30%. Also, unintended fragments produced by nonhomologous end-joining were observed between the cleavage site at the terminus of the donor DNA in the AdV genome and the target site in the cell genome. CONCLUSIONS:The knock-in efficiency of AdVs can be increased by cleaving the terminus of the donor DNA, although it would be desirable to avoid nonhomologous end-joining between double-strand break termini.
BACKGROUND:Myocardial ischemia-reperfusion injury (MIRI) remains a major clinical problem, and its regulatory mechanisms are not fully defined. This study aimed to identify key MIRI-related targets and clarify their roles in cellular and animal models. METHODS:Differentially expressed genes in MIRI were screened using the GEO dataset GSE6381, and core genes were identified through protein-protein interaction analysis and weighted gene co-expression network analysis. External datasets GSE249812 and GSE123342 were used to validate core gene expression. Transcription factors regulating cystic fibrosis transmembrane conductance regulator (CFTR) were predicted using the KnockTF database. In AC16 cells, an oxygen-glucose deprivation/reoxygenation (OGD/OGR) model was established. Microphthalmia-associated transcription factor (MITF) was knocked down or overexpressed, CFTR was silenced or overexpressed, and cell viability, proliferation, apoptosis, and NO/L-arginine/citrulline levels were assessed. The MITF-CFTR interaction was examined by dual-luciferase assay and chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR). A rat ischemia-reperfusion (I/R) model was used to evaluate the effects of MITF silencing and CFTR rescue on cardiac injury. RESULTS:A total of 42 core genes related to MIRI were identified. CFTR showed consistent upregulation across datasets and was linked to pathways including arginine biosynthesis. CFTR-related transcription factors included MITF, TP53, and STAT3, but ChIP-qPCR showed detectable enrichment only for MITF at the CFTR promoter. In AC16 cells, CFTR protein increased during early reoxygenation. OGD/OGR elevated CFTR and MITF, while MITF knockdown or CFTR silencing further reduced cell viability and proliferation, increased apoptosis, and was accompanied by lower NO and citrulline levels and higher L-arginine levels. MITF overexpression showed the opposite trend, and CFTR overexpression partially reversed the effects of MITF deficiency. In vivo, MITF knockdown aggravated cardiac dysfunction, increased CK and CK-MB levels, worsened tissue injury, and elevated cleaved caspase-3 after I/R, whereas CFTR overexpression partly improved these changes. CONCLUSIONS:MITF regulates CFTR expression in MIRI, and disruption of the MITF-CFTR axis is associated with aggravated injury in cellular and animal models. Restoring CFTR partially mitigates MITF deficiency-related damage. These findings support the involvement of MITF-CFTR regulation in MIRI and provide a basis for further mechanistic investigation.
OBJECTIVE:Mesenchymal stem cell exosomes (MSC-Exos) play crucial regulatory roles in the processes of alveolar epithelial cell development, proliferation, apoptosis and differentiation. The aim of this project was to explore the regulatory mechanism of the miR-23a-3p/Slc7a2 axis mediated by bone marrow MSC-derived exosomes in hyperoxia-induced injury to alveolar epithelial type II cells (AECIIs) in neonatal rats. METHODS:The miRBase and TargetScan databases were used to intersect and validate the differential expressions of microRNAs derived from BMSC exosomes in AECIIs under hyperoxic conditions and their target genes to construct in vitro and in vivo models of lung epithelial cell injury under hyperoxic conditions and to transfect BMSC-Exos. Dual-luciferase reporter assays were used to verify the binding relationship between miR-23a-3p and Slc7a2 and overexpression and siRNA plasmids for miR-23a-3p and Slc7a2 were constructed. RESULTS:miR-23a-3p derived from BMSC exosomes directly targets Slc7a2 mRNA. miR-23a-3p antagonizes hyperoxia-induced apoptosis of AECIIs and promotes cell proliferation in vitro by inhibiting Slc7a2 expression (p < 0.05). miR-23a-3p inhibited, while Slc7a2 promoted, the expression of ROS in AECIIs. miR-23a-3p significantly ameliorated hyperoxia-induced lung tissue injury in neonatal rats and significantly reduced hyperoxia-induced apoptosis in neonatal rat lung tissue (p < 0.05). The miR-23a-3p/Slc7a2 axis significantly regulates the expression of multiple biochemical markers in the supernatant of AECIIs and in the serum of neonatal rats under hyperoxia conditions. CONCLUSION:miR-23a-3p derived from BMSC exosomes participates in the repair of lung epithelial cell injury under hyperoxic conditions by regulating the expression of its target gene Slc7a2.
Background Dysregulation of transcription factors potentiates cancer cell proliferation, stemness, cellular plasticity, metastasis, and therapy resistance and also links with diagnosis/prognosis of the diseases. Thus, investigation of uncharacterized transcription factors is a prime aim for designing novel therapeutics. Large family C2H2 type zinc finger proteins (ZNFs) often bind to nucleic acids and also act as transcription factors. However, unregulated expression of these ZNFs was found to cause diverse pathological conditions including cancer.Methods Cancer database and specific GEO analysis were used to screen unexplored ZNFs. Subsequent knockdown and overexpression studies of selected ZNFs were conducted to examine their oncogenicity in breast cancer cells. Database analysis, ChIP, and knockdown study identified target genes. Overexpression and rescue experiments identified the oncogenic potential of target genes. The chemo-sensitivity of the target gene was determined. RT-qPCR analysis of breast cancer tissues confirms the oncogenic potential of the ZNFs.Results A systematic cancer database analysis using differential gene expressions (fold change between tumor and control tissue) and patient survivability (hazard ratio) found three unexplored ZNFs in breast cancer. Subsequent GEO database analysis determined ZNF468 for further experimentation. Knockdown of the gene ZNF468 showed inhibition of various oncogenic potentials including cell proliferation, migration, invasion, and epithelial to mesenchymal transition (EMT) and oncogenic markers (e.g., Bcl-2, Vimentin, and Zeb2) in both MCF-7 and MDA-MB-231 breast cancer cells. Database analysis found consensus DNA binding site of the ZNF468 in unexplored gene ZNF707, further confirmed by ChIP assay and knockdown of ZNF468. Subsequently, overexpression of ZNF707 gene upregulated cell proliferation, migration, invasion, and oncogenic markers in both MCF-7 and MDA-MB-231 cells. ZNF468 knockdown also inhibited the expression of various cholesterol regulatory genes. Furthermore, the oncogenic activity of ZNF468 in MCF-7 cells was revived by the overexpression of ZNF707. Overexpression of ZNF707 reduced the effectiveness of doxorubicin treatment. Finally, compared to benign, expression of both ZNF468 and ZNF707 was higher in malignant breast cancer patient tissue.Conclusions These findings for the first time documented the oncogenic potential of ZNF707 in breast cancer. Additionally, ZNF468 promotes its oncogenic activity by regulating ZNF707 expression. Thus, these two ZNFs may be further explored to design promising therapy for breast cancer treatment.
BACKGROUND/AIM:Recurrent miscarriage (RM) is a major reproductive concern affecting a large number of women worldwide. Genetic factors are thought to play an important role in RM. Due to its potential function in angiogenesis, the histidine-rich glycoprotein (HRG) gene has become a focus of research. Therefore, the present study aimed to determine the association between the HRG A1043G single-nucleotide polymorphism (SNP) and RM. MATERIALS AND METHODS:To meet the objectives of the study, 110 women with a history of RM were included. The control group consisted of 60 women who had delivered children and had no history of miscarriage. Blood samples were collected from all participants; genomic DNA was extracted and amplified using polymerase chain reaction (PCR). Sanger sequencing was performed to screen for the A1043G polymorphism in exon 7 of the HRG gene. RESULTS:The findings revealed a significant association between the HRG SNP and the risk of RM at both genotypic and allelic levels. Compared to the AA genotype, mutant genotypes (AG + GG) showed a significantly higher risk of RM (p = 0.041). The G allele was also significantly associated with RM, as indicated by the odds ratio (OR) analysis (p = 0.018). CONCLUSIONS:The present study demonstrated that genetic variation in the HRG gene may contribute to susceptibility to RM.
GNE myopathy is an autosomal recessive disease, associated with skeletal muscle deterioration, which afflicts young adults. GNE plays a pivotal role in sialic acid production. Sialic acid acts as a buffer against reactive oxygen species generated during muscle contraction. Increased oxidative stress may relate to muscle atrophy involving patients with GNE myopathy. GNEM743T is the most common mutation leading to GNE myopathy. In our previous work, we demonstrated that a bifunctional plasmid that expresses wild type (wt) GNE and knocks down the GNEM743T mutant improves sialic acid production in vitro. Now, we expand evidence of in vivo activity of the bifunctional plasmid using a DOTAP-Cholesterol delivery vehicle and reduced toxic plasmid components using dbDNA conversion. We demonstrate that IV delivery of dbDNA lipoplex (LPX) in murine and rat models shows safety, increased DNA delivery, and improved RNA expression per LPX in skeletal muscle over non db plasmid at equal dose. Sialic acid protein expression was also shown increased in mouse muscle following IV treatment with dbDNA plasmid (pDNA) GNEwt/bi-shRNA-GNEM743T LPX. These results support further preclinical investigation to justify product IND development towards Phase 1 trial involving patients with GNE myopathy.
BACKGROUND:In males, prostate cancer (PCa) is one of the frequently diagnosed forms of cancer, with high clinical variability and limited treatment options for advanced cases. This receptor, which goes by the names Coagulation Factor II Receptor (F2R) and PAR1, belongs to the family of G-protein-linked membrane proteins and plays roles in both blood clotting processes and the development of malignancies. Whereas F2R has been associated with tumor progression in various malignancies, its specific involvement in PCa is not well understood. Here, we seek to examine the expression patterns and biological functions of F2R to better understand its impact on PCa progression. METHODS:We systematically analyzed F2R expression in PCa using data from the TCGA database and clinical specimens. Functional experiments, including cell proliferation, invasion, and apoptosis assays, were conducted in PCa cell lines with F2R overexpression or knockdown. Bioinformatics analyses were performed to identify F2R-associated genes and signaling pathways. In vivo xenograft models were used to validate the oncogenic role of F2R. RESULTS:Our results demonstrated that F2R is significantly overexpressed in PCa tissues and correlates with advanced clinicopathological features such as higher T stage, nodal metastasis, and elevated Gleason scores. Functional studies revealed that F2R promotes PCa cell proliferation, invasion, and cell cycle progression while inhibiting apoptosis. Mechanistically, we identified collagen type VIII alpha 1 (COL8A1) as a key downstream effector of F2R, which activates the FAK/PI3K/AKT signaling pathway. In vivo experiments confirmed that F2R knockdown suppresses tumor growth and downregulates this signaling axis. CONCLUSION:This study highlights F2R as an important promoter in PCa progression and identifies the F2R-COL8A1-FAK/PI3K/AKT signaling axis as a potential molecular mechanism underlying tumor aggressiveness.
Objective This study aimed to explore whether CD74 participates in regulating ferroptosis and to clarify the related mechanisms in traumatic brain injury (TBI).Methods A TBI rat model was generated using controlled cortical impact. The ferroptosis inducer RSL-3, the inhibitor Liproxstatin-1 (Lip-1), and lentiviral vectors targeting CD74 or Nrf2 were injected into the lateral ventricle. Knockdown efficiency of the lentiviral vectors was verified by RT-qPCR. Motor performance was evaluated using the foot fault test, neurobehavioral function via mNSS scoring, brain water content using the wet-dry method, iron deposition in cortical tissues by Perls' Blue staining, Fe2+ levels with an iron assay kit, degenerating neurons by Fluoro-Jade C staining, and Nrf2/HO-1 pathway protein expression via Western blot.Results TBI rats displayed increased foot faults, elevated mNSS scores, increased brain water content, higher Fe2+ levels, more iron-positive cells, and greater numbers of degenerating neurons in the cerebral cortex. Lip-1 or CD74 downregulation alleviated TBI-related changes, whereas RSL-3 or CD74 upregulation worsened them. Downregulating CD74 enhanced Nrf2/HO-1 pathway activity, and Nrf2 knockdown counteracted the benefits of CD74 downregulation.Conclusion Reducing CD74 expression ameliorates ferroptosis in TBI by activating the Nrf2/HO-1 signaling axis.
BACKGROUND:The SGLT2 inhibitor empagliflozin (EMPA) has been found to reduce the combined risk of cardiovascular death or hospitalization for heart failure in patients with or without reduced left ventricular ejection fraction, irrespective of diabetes status. The underlying mechanisms remain to be elucidated. Endothelial-to-mesenchymal transition (EndoMT) has been reported to play a pivotal role in the microvascular rarefaction. This study aimed to evaluate the effect of EMPA on angiotensin II (Ang II)-induced left ventricular dysfunction and to explore the underlying mechanism. METHODS:In vivo, C57BL/6J mice were infused with saline or Ang II (1.5 mg/kg/day) and subsequently treated with or without EMPA (10 mg/kg) for 2 weeks. mRNA sequencing and gene set enrichment analysis (GSEA) indicated that the PI3K/AKT/eNOS signalling pathway may mediate the protective effects of empagliflozin in heart failure with preserved ejection fraction (HFpEF). Finally, in vitro, PI-103 was used to treat cells, and immunofluorescence, western blotting, qPCR, and other methods were used to verify whether empagliflozin exerts its effects through the PI3K/AKT/eNOS pathway. RESULTS:In vivo, the mice treated with Ang II exhibited left ventricular dysfunction, increased microvascular rarefaction, and EndoMT, all of which were attenuated by EMPA treatment. In vitro, primary cardiac microvascular endothelial cells (CMECs) exposed to Ang II showed increased EndoMT, which was significantly inhibited by EMPA. EMPA also reversed the downregulation of PI3K/AKT/eNOS signalling and nitric oxide (NO) levels. PI-103 abrogated the anti-EndoMT effects of EMPA in CMECs. CONCLUSIONS:Our study suggested that EMPA can protect against Ang II-induced left ventricular dysfunction and microvascular rarefaction by suppressing EndoMT via PI3K/AKT/eNOS signalling.
BACKGROUND:Pneumonia caused by Klebsiella pneumonia (Kp) poses a significant risk to global public health. Vitamin D may reduce Kp infection risk and improve prognosis through immunomodulation. This study aimed to validate the treatment effects of Vitamin D and explore its regulatory mechanism in Kp-pneumonia. METHODS:In this study, a murine model of Kp-induced pneumonia and the MH-S alveolar macrophage cell line were used. Experimental assays included RT-qPCR, Western blot, TUNEL assay, ELISA, flow cytometry, dual-luciferase reporter assay, and metabolic analyses (FAO activity, Seahorse XF Glycolysis Stress Test). RESULTS:The results showed that vitamin D administration mitigated Kp-induced lung injury in mice. Mechanically, vitamin D alleviated inflammation by inhibiting macrophage M1 polarization. Vitamin D exerted its effects by upregulating miR-223, which directly targeted and suppressed ACSL3 expression. In macrophages, miR-223 overexpression alleviated macrophage apoptosis and M1 polarization by downregulating ACSL3. Knockdown of ACSL3 induced a shift to M2 polarization by enhancing FAO and suppressing glycolysis. In vivo, miR-223 overexpression alleviated Kp-induced lung injury by downregulating ACSL3. CONCLUSION:In conclusion, vitamin D induces macrophage M2 polarization by upregulating miR-223, which inhibits ACSL3, leading to lipid metabolism reprogramming. This novel axis represents a potential therapeutic strategy for Kp-induced pneumonia.
CRISPR-Cas systems have emerged as versatile platforms for targeted genome and transcriptome engineering, enabling precise manipulation of disease-associated genetic pathways. Continued advances in CRISPR technologies including base editing, prime editing, and epigenome modulation have expanded therapeutic possibilities beyond nuclease-mediated DNA cleavage, allowing programmable gene correction and regulation. Early clinical studies demonstrate sustained therapeutic benefit in selected monogenic disorders and highlight the feasibility of both ex vivo and in vivo editing strategies. However, clinical translation remains constrained by challenges such as off-target activity, delivery inefficiency, immune responses to Cas proteins, editing heterogeneity, and uncertainties regarding long-term safety. This review critically synthesizes recent advances in CRISPR-Cas systems for human disease therapy, integrating molecular innovations, delivery strategies, clinical progress, and ethical considerations. By evaluating both technological achievements and unresolved limitations, this article outlines key priorities for advancing CRISPR-based therapeutics toward safe, effective, and equitable precision medicine.
Background Neurodevelopmental disorders (NDDs) are clinically heterogeneous conditions with complex etiologies and limited therapeutic options. Here, we investigated a proband presenting global developmental delay (GDD), tonic seizures, failure to thrive, mild microcephaly, intellectual disability (ID), and hypotonia. Methods Exome sequencing (ES), followed by Sanger sequencing, was performed for molecular diagnosis. Gene expression was assessed by reverse-transcriptase quantitative PCR (RT-qPCR), and 3D protein modeling was performed. Results ES revealed a novel de novo (heterozygous) missense variant [c.2660C>T; p.(Pro887Leu)], in LRRC7 (NM_001370785.2) gene, located in exon 18, which may contribute to the proband's phenotype. The identified variant was classified as variant of uncertain significant (VUS) according to the American College of Medical Genetics and Genomics Guidelines (ACMG). RT-qPCR showed reduced LRRC7 mRNA expression in the proband compared to control samples, while and 3D protein modeling revealed substantial changes in the LRRC7-secondary structure. Conclusion Using genetic, molecular, in silico, and expression analysis, we characterize a novel de novo-LRRC7 variant and describe its association with an NDD phenotype.
Nonviral gene therapy holds promise as a potential treatment for lung cystic fibrosis (CF). However, the transient expression of the CF transmembrane conductance regulator (CFTR) protein has limited its clinical application. To circumvent this challenge, a CpG-depleted CFTR plasmid was developed. The CpG-depleted CFTR plasmid could be compacted into DNA nanoparticles and modified with the addition of highly branched poly(β-amino ester)s (HPAEs), leading to an improved and sustained CFTR protein expression. Using a CpG-depleted and codon-optimized CFTR sequence, around 20-fold increase in CFTR protein production was achieved 48 h after treatment, compared with healthy human bronchial epithelial cells (16HBE14o-). To evaluate the duration of CFTR protein expression induced by the plasmid based on human elongation factor 1α (hEFIα) and cytomegalovirus (CMV) promoters, a time course study was conducted in human CF bronchial epithelial (CFBE14o-) cells. hEFIα promoter revealed a remarkable 2.26-fold increase in CFTR protein expression at 7 days after transfection compared with 16HBE14o- cells. This level of CFTR protein expression outperformed the commonly used CMV promoter. The in vitro results demonstrated that CpG-depleted CFTR plasmid could be used to achieve high efficacy in subsequent in vivo evaluations, which, if validated, may provide promising prospects for the development of a novel and effective treatment for lung cystic fibrosis.
Background Osteoarthritis (OA) is a chronic degenerative joint disease characterized by the progressive deterioration of articular cartilage, significantly impacting the quality of life in middle-aged and elderly populations. The Ras and Hippo signaling pathways play critical roles in regulating cell proliferation, differentiation, and stress responses; however, their interactive mechanisms in OA remain unclear. This study aimed to identify key genes associated with these two pathways using bioinformatic approaches and to elucidate their potential mechanisms in OA.Methods Transcriptomic data of OA along with Ras signaling pathway-related genes (RSPRGs) and Hippo signaling pathway-related genes (HSPRGs) were obtained from public databases. Differentially expressed genes (DEGs) were identified, and key genes were screened through machine learning, expression validation, and receiver operating characteristic (ROC) curve analysis. Functional insights were further explored via gene set enrichment analysis (GSEA), subcellular localization, immune infiltration analysis, regulatory network construction, and drug prediction. Finally, the expression of key genes was validated in clinical samples.Results KIT and CSF1R were identified as key genes. GSEA indicated their involvement in pathways such as the lysosome pathway. Subcellular localization predicted that KIT and CSF1R are distributed in the nucleus, extracellular region, and plasma membrane. Immune infiltration analysis revealed that KIT showed a positive correlation with eosinophils and a negative correlation with immature dendritic cells, whereas CSF1R was positively correlated with macrophages and negatively correlated with CD56bright natural killer cells. Drug prediction suggested interactions between the key genes and several therapeutic agents, including avapritinib and IMC-CS4. Subsequently, we validated our findings in cartilage tissue samples and discovered that compared to the control group, both CSF1R mRNA and protein expression were significantly upregulated in OA tissue, while KIT expression was significantly downregulated. The same were also validated in immunofluorescence staining of chondrocytes.Conclusion This study identified KIT and CSF1R as key genes in OA, providing new theoretical insights and potential targets for mechanistic research and targeted therapy.
Chronic granulomatous disease (CGD) is a rare inborn error of immunity caused by defects in components of the NADPH oxidase that impair the elimination of infectious microorganisms. Individuals affected by CGD become more susceptible to recurrent and severe infections. Six male patients from Southern Brazil were clinically and genetically analyzed through data collection from medical records and massively parallel sequencing by a panel for the following genes: CYBB, CYBA, NCF1, NCF2, and NCF4 and whole genome sequencing analysis. The gene-scan technique was used to identify the GT deletion in NCF1. The most common affected organs were the lungs, skin, and lymph nodes; the most common clinical manifestations were recurrent pneumonia, cutaneous involvement, lymph node manifestations, and failure to thrive. Four patients were identified with variants in CYBB: p.Cys257Ser, which is novel; p.Cys257Arg; p.Arg157Ter; and p.Trp483Ter. Both missense variants damage the loop E in gp91phox, a region with functional and structural relevance for the protein. Functional studies show the expression absence of the protein in patients with the variant p.Arg157Ter. The variant p.Trp483Ter is predicted to undergo nonsense mRNA-mediated decay. The GT deletion in NCF1 was identified in two siblings from consanguineous parents: one homozygous and the other apparently heterozygous for the deletion, both with a clinical diagnosis of CGD. Variant analysis in this gene is particularly challenging due to the presence of pseudogenes. A hypothesis for this genotypic discrepancy is the occurrence of a second type of pseudogene lacking the GT deletion, which may have arisen in one parent and been transmitted to the patient observed as heterozygous, being misinterpreted in the analyses as a functional NCF1 sequence.
BACKGROUND:Chronic tendon injuries, characterized by persistent pain, reduced flexibility, and impaired function, pose a significant clinical challenge. Current therapeutic strategies for these injuries are limited. This study highlighted the crucial role of OXPHOS in maintaining tendon homeostasis and suggested potential therapeutic strategies targeting the OXPHOS pathway. METHOD:This study utilized both bulk-sequencing (bulk-seq) and single-cell RNA sequencing (scRNA-seq) to analyze the heterogeneity in tenocytes, vascular endothelial cells, tendon-derived stem cells, adipocytes, and neurons from both non-lesional and lesional tendons. Key oxidative phosphorylation (OXPHOS)-related genes, such as COX15, COX4I1, COX5B, COX7A1, COX8A, NDUFA12, NDUFA5, NDUFB10, NDUFB3, NDUFC1, NDUFS1, and NDUFS4, were found to be significantly downregulated in lesional tendons compared with non-lesional ones, indicating impaired energy metabolism. This reduction in OXPHOS activity may contribute to increased necroptosis in chronic tendon injuries. Furthermore, bisphenol A and valproic acid were found to activate OXPHOS-related genes. RESULTS:The findings highlighted the crucial role of OXPHOS in maintaining tendon homeostasis and demonstrated potential therapeutic strategies targeting the OXPHOS pathway, such as bisphenol A and valproic acid, to enhance healing in chronic tendon conditions. CONCLUSION:The crucial role of OXPHOS in maintaining tendon homeostasis underscores its potential as a therapeutic target, reflecting that strategies aimed at modulating the OXPHOS pathway may provide promising treatment options. Chronic tendon injuries present a major clinical challenge with limited treatments. This study investigated the molecular mechanisms underlying these injuries using bulk and single-cell RNA sequencing. We identified significant downregulation of oxidative phosphorylation (OXPHOS)-related genes in lesional tendons across multiple cell types, contributing to increased necroptosis. In vitro experiments and molecular docking revealed that valproic acid activates OXPHOS and inhibits necroptosis. These findings highlight the critical role of mitochondrial function in tendon homeostasis and suggest valproic acid as a promising therapeutic candidate for treating chronic tendon injuries by restoring OXPHOS activity.
Ovarian cancer remains a leading cause of gynecological malignancy-related deaths, necessitating the identification of novel molecular pathways driving tumor progression. Utilizing a data-driven approach, we conducted bioinformatic analyses of TCGA and GEO datasets, identifying a significant upregulation of CXCL14 in ovarian cancer tissues, which correlates with poor patient survival. Functional assays demonstrated that overexpression of CXCL14 enhances ovarian cancer cell proliferation, invasion, and autophagy. Mechanistically, CXCL14 activates the canonical NF-κB signaling pathway by inducing phosphorylation and degradation of IκBα, leading to phosphorylation and nuclear translocation of p65. Importantly, we identified IKBKE as a critical kinase mediating CXCL14-induced activation of the canonical NF-κB pathway through phosphorylation of IκBα. Knockdown of IKBKE effectively attenuates CXCL14-driven NF-κB activation, thereby suppressing cell proliferation, invasion, and autophagy. In vivo, CXCL14 overexpression markedly enhances ovarian tumor growth, accompanied by increased levels of IKBKE and phosphorylated p65. These findings elucidate a novel regulatory axis, CXCL14/IKBKE/NF-κB, in ovarian cancer progression, highlighting CXCL14 as a potential therapeutic target for ovarian cancer treatment.
