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Objective: This study is aimed at investigating diagnostic biomarkers associated with lipotoxicity and the molecular mechanisms underlying diabetic nephropathy (DN). Methods: The GSE96804 dataset from the Gene Expression Omnibus (GEO) database was utilized to identify differentially expressed genes (DEGs) in DN patients. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were conducted using the DEGs. A protein-protein interaction (PPI) network was established to identify key genes linked to lipotoxicity in DN. Immune infiltration analysis was employed to identify immune cells with differential expression in DN and to assess the correlation between these immune cells and lipotoxicity-related hub genes. The findings were validated using the external dataset GSE104954. ROC analysis was performed to assess the diagnostic performance of the hub genes. The Gene set enrichment analysis (GSEA) enrichment method was utilized to analyze the key genes associated with lipotoxicity as mentioned above. Result: In this study, a total of 544 DEGs were identified. Among them, extracellular matrix (ECM), fatty acid metabolism, AGE-RAGE, and PI3K-Akt signaling pathways were significantly enriched. Combining the PPI network and lipotoxicity-related genes (LRGS), LUM and ALB were identified as lipotoxicity-related diagnostic biomarkers for DN. ROC analysis showed that the AUC values for LUM and ALB were 0.882 and 0.885, respectively. The AUC values for LUM and ALB validated in external datasets were 0.98 and 0.82, respectively. Immune infiltration analysis revealed significant changes in various immune cells during disease progression. Macrophages M2, mast cells activated, and neutrophils were significantly associated with all lipotoxicity-related hub genes. These key genes were enriched in fatty acid metabolism and extracellular matrix-related pathways. Conclusion: The identified lipotoxicity-related hub genes provide a deeper understanding of the development mechanisms of DN, potentially offering new theoretical foundations for the development of diagnostic biomarkers and therapeutic targets related to lipotoxicity in DN.
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Biomarcadores , Biología Computacional , Nefropatías Diabéticas , Perfilación de la Expresión Génica , Mapas de Interacción de Proteínas , Humanos , Nefropatías Diabéticas/genética , Nefropatías Diabéticas/metabolismo , Nefropatías Diabéticas/diagnóstico , Biomarcadores/metabolismo , Lumican/genética , Lumican/metabolismo , Ontología de Genes , Redes Reguladoras de Genes , Bases de Datos Genéticas , Transducción de SeñalRESUMEN
PURPOSE: The recent focus on the roles of N-linked glycoproteins in carcinogenesis across various malignancies has prompted our exploration of aberrantly expressed glycoproteins responsible for HCC progression and potential therapeutic strategy. METHODS: Mass spectrometry was applied to initially identify abnormally expressed glycoproteins in HCC, which was further assessed by immunohistochemistry (IHC) staining. The role of selected glycoprotein on HCC development and underlying mechanism was systematically investigated by colony formation, mouse xenograft, RNA-sequencing and western blot assays, etc. Chromatin immunoprecipitation (ChIP) and luciferase assays were performed to explore potential transcription factors (TFs) of selected glycoprotein. The regulation of repaglinide (RPG) on expression of lumican and downstream effectors was assessed by western blot and IHC, while its impact on malignant phenotypes of HCC was explored through in vitro and in vivo analyses, including a murine NASH-HCC model established using western diet and carbon tetrachloride (CCl4). RESULTS: Lumican exhibited upregulation in both serum and tumor tissue, with elevated expression associated with an inferior prognosis in HCC patients. Knockdown of lumican resulted in significantly reduced growth of HCC in vitro and in vivo. Mechanically, lumican promoted HCC malignant phenotypes by inhibiting the p53/p21 signaling pathway. Forkhead Box O3 (FOXO3) was identified as the TF of lumican that transcriptionally enhanced its expression. Without silencing FOXO3, RPG blocked the binding of FOXO3 to the promoter region of lumican, thereby inhibiting the activation of lumican/p53/p21 axis. Mice treated with RPG developed fewer and smaller HCCs than those in the control group at 24 weeks after establishment. CONCLUSION: Our results indicate that RPG prevented the development and progression of HCC via alteration of FOXO3/lumican/p53 axis.
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Carcinoma Hepatocelular , Progresión de la Enfermedad , Proteína Forkhead Box O3 , Neoplasias Hepáticas , Lumican , Transducción de Señal , Proteína p53 Supresora de Tumor , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Animales , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/genética , Proteína Forkhead Box O3/metabolismo , Proteína Forkhead Box O3/genética , Proteína p53 Supresora de Tumor/metabolismo , Proteína p53 Supresora de Tumor/genética , Ratones , Transducción de Señal/efectos de los fármacos , Lumican/metabolismo , Lumican/genética , Línea Celular Tumoral , Masculino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Femenino , Persona de Mediana Edad , Ratones Desnudos , Carbamatos , PiperidinasRESUMEN
OBJECTIVES: The past studies clearly indicated that lumican was important in the context of pancreatic cancer (PC) onset and progression, but failed to clarify the underlying mechanistic basis for such activity. As such, we evaluated the functional importance of lumican in the context of pancreatic ductal adenocarcinoma (PDAC) to understand its mechanistic role in PC. METHODS: Lumican levels were evaluated in PDAC patient tissues via quantitative real-time polymerase chain reaction, Western blotting, and immunohistochemistry approaches. The role of lumican was additionally assessed via transfecting PDAC cell lines (BxPC-3, PANC-1) with lumican knockdown or overexpression constructs and treating PDAC cell lines with exogenous recombinant human lumican. RESULTS: Lumican expression levels were significantly higher in pancreatic tumor tissues relative to healthy paracancerous tissues. Lumican knockdown in BxPC-3 and PANC-1 enhanced their proliferation and migration, but reduced cellular apoptosis. Alternatively, lumican overexpression and exogenous lumican exposure failed to alter the proliferative activity of these cells. Further, lumican knockdown in BxPC-3 and PANC-1 cells results in marked P53 and P21 dysregulation. CONCLUSIONS: Lumican may suppress PDAC tumor growth by regulating P53 and P21, and the function of lumican sugar chains in the context of PC is worth studying in future studies.
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Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Lumican/genética , Lumican/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Neoplasias Pancreáticas/patología , Páncreas/patología , Carcinoma Ductal Pancreático/patología , Hormonas Pancreáticas/metabolismo , Proliferación Celular/genética , Línea Celular Tumoral , Movimiento Celular , Regulación Neoplásica de la Expresión Génica , Neoplasias PancreáticasRESUMEN
Abnormal expression of p120 catenin is associated with the malignant phenotype in human lung cancer. Numerous studies have focused on the function of p120 catenin in the juxta-membrane compartment. However, the role of nuclear p120 catenin remains unclear. In this study, the dynamic changes in nuclear p120 catenin localization during cell cycle progression were investigated. Immunofluorescent staining, FACS analysis, and western blotting revealed that nuclear p120 catenin is a major architectural constituent of the chromosome periphery during mitosis. During mitosis, granule-like p120 catenin dispersed into a cloudy-like structure and formed cordon-like structures surrounding the condensed chromosomes to create the peri-chromosomal layer. Interestingly, lumican and p120 catenin colocalized at the spindle fiber where the perichromosomal layer connects to the condensed chromosomes during mitosis. Furthermore, downregulation of p120 catenin using a specific siRNA induced cell cycle stalling in the G2/M phase and promoted aneuploidy. This study validates the role of nuclear p120 catenin in the formation of the chromosome periphery and reveals the p120 catenin-lumican interaction may couple orientation of cell division with the segregation of sister chromatids during mitosis. Our data suggest the protective role of p120 catenin in maintaining the integrity of chromosomes, and also warrants further studies to evaluate the contribution of the loss of p120 catenin to the creation of gene rearrangement in cancer evolution and tumor progression.
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Cromátides , Neoplasias Pulmonares , Cateninas/metabolismo , Cromátides/genética , Cromátides/metabolismo , Cromátides/patología , Segregación Cromosómica , Humanos , Lumican/genética , Neoplasias Pulmonares/patología , Mitosis/genéticaRESUMEN
The cervix undergoes rapid and dramatic shifts in collagen and elastic fiber structure to achieve its disparate physiological roles of competence during pregnancy and compliance during birth. An understanding of the structure-function relationships of collagen and elastic fibers to maintain extracellular matrix (ECM) homeostasis requires an understanding of the mechanisms executed by non-structural ECM molecules. Small-leucine rich proteoglycans (SLRPs) play key functions in biology by affecting collagen fibrillogenesis and regulating enzyme and growth factor bioactivities. In the current study, we evaluated collagen and elastic fiber structure-function relationships in mouse cervices using mice with genetic ablation of decorin and/or biglycan genes as representative of Class I SLRPs, and lumican gene representative of Class II SLRP. We identified structural defects in collagen fibril and elastic fiber organization in nonpregnant mice lacking decorin, or biglycan or lumican with variable resolution of defects noted during pregnancy. The severity of collagen and elastic fiber defects was greater in nonpregnant mice lacking both decorin and biglycan and defects were maintained throughout pregnancy. Loss of biglycan alone reduced tissue extensibility in nonpregnant mice while loss of both decorin and biglycan manifested in decreased rupture stretch in late pregnancy. Collagen cross-link density was similar in the Class I SLRP null mice as compared to wild-type nonpregnant and pregnant controls. A broader range in collagen fibril diameter along with an increase in mean fibril spacing was observed in the mutant mice compared to wild-type controls. Collectively, these findings uncover functional redundancy and hierarchical roles of Class I and Class II SLRPs as key regulators of cervical ECM remodeling in pregnancy. These results expand our understating of the critical role SLRPs play to maintain ECM homeostasis in the cervix.
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Proteoglicanos Pequeños Ricos en Leucina , Neoplasias del Cuello Uterino , Animales , Biglicano/genética , Biglicano/metabolismo , Cuello del Útero/metabolismo , Proteoglicanos Tipo Condroitín Sulfato/genética , Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Decorina/genética , Decorina/metabolismo , Proteínas de la Matriz Extracelular/genética , Femenino , Fibromodulina , Humanos , Lumican/genética , Ratones , Embarazo , Proteoglicanos Pequeños Ricos en Leucina/genéticaRESUMEN
Fibrogenic and inflammatory processes in the prostate are linked to the development of lower urinary tract symptoms (LUTS) in men. Our previous studies identified that osteopontin (OPN), a pro-fibrotic cytokine, is abundant in the prostate of men with LUTS, and its secretion is stimulated by inflammatory cytokines potentially to drive fibrosis. This study investigates whether the lack of OPN ameliorates inflammation and fibrosis in the mouse prostate. We instilled uropathogenic E. coli (UTI89) or saline (control) transurethrally to C57BL/6J (WT) or Spp1tm1Blh/J (OPN-KO) mice and collected the prostates one or 8 weeks later. We found that OPN mRNA and protein expression were significantly induced by E. coli-instillation in the dorsal prostate (DP) after one week in WT mice. Deficiency in OPN expression led to decreased inflammation and fibrosis and the prevention of urinary dysfunction after 8 weeks. RNAseq analysis identified that E. coli-instilled WT mice expressed increased levels of inflammatory and fibrotic marker RNAs compared to OPN-KO mice including Col3a1, Dpt, Lum and Mmp3 which were confirmed by RNAscope. Our results indicate that OPN is induced by inflammation and prolongs the inflammatory state; genetic blockade of OPN accelerates recovery after inflammation, including a resolution of prostate fibrosis.
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Infecciones por Escherichia coli/genética , Osteopontina/genética , Próstata/metabolismo , Infecciones Urinarias/genética , Escherichia coli Uropatógena/patogenicidad , Animales , Proteoglicanos Tipo Condroitín Sulfato/genética , Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Colágeno Tipo III/genética , Colágeno Tipo III/metabolismo , Modelos Animales de Enfermedad , Infecciones por Escherichia coli/metabolismo , Infecciones por Escherichia coli/patología , Infecciones por Escherichia coli/prevención & control , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Fibrosis , Regulación de la Expresión Génica , Humanos , Inflamación , Lumican/genética , Lumican/metabolismo , Masculino , Metaloproteinasa 3 de la Matriz/genética , Metaloproteinasa 3 de la Matriz/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Osteopontina/deficiencia , Próstata/patología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de Señal , Infecciones Urinarias/metabolismo , Infecciones Urinarias/patología , Infecciones Urinarias/prevención & control , Escherichia coli Uropatógena/crecimiento & desarrolloRESUMEN
A recent study has reported that lumican (LUM) is expressed at a high level in the nucleus pulposus specimens from herniated lumbar disc, without description of the specific mechanism. This study was designed to investigate the function and mechanism of LUM in intervertebral disc degeneration (IDD). In this study, human nucleus pulposus cells (hNPCs) cells were challenged with tumor necrosis factor (TNF)-α to establish the IDD in vitro model. After LUM silencing, cell viability was detected using CCK-8 kit, and the expression of inflammatory factors was evaluated using RT-qPCR and ELISA. Flow cytometry and ß-galactosidase staining were used to determine cell cycle and cell senescence. The expression of cycle and senescence-related proteins was evaluated with western blotting. Then, Fas ligand (FasL) was overexpressed and proteins in apoptosis signal regulating kinase 1 (ASK1)/p38 signaling were tested. Finally, GS-4997, an inhibitor of ASK1, was used to explore the regulatory effects of LUM on ASK1/p38 signaling in TNF-α-induced hNPCs. Results indicated that LUM expression was upregulated in TNF-α-challenged hNPCs. LUM gene interference mitigated TNF-α-induced inflammatory response, cell cycle arrest, and senescence of hNPCs. It was then found that LUM silencing could inhibit ASK1/p38 signaling in TNF-α-treated hNPCs, which was reversed by FasL overexpression. Additionally, ASK1/p38 participated in the mediation by LUM of TNF-α-induced inflammation, cell cycle arrest, and senescence of hNPCs. To conclude, interference with LUM effectively mitigated TNF-α-induced inflammatory response, cell cycle arrest, and cell senescence. Further experiments showed the involvement of ASK1/p38 pathway in LUM-mediated NP cell phenotypes through FasL.
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Senescencia Celular/genética , Lumican , MAP Quinasa Quinasa Quinasa 5/genética , Núcleo Pulposo/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Ciclo Celular/genética , Células Cultivadas , Proteína Ligando Fas/genética , Proteína Ligando Fas/metabolismo , Silenciador del Gen , Humanos , Inflamación/genética , Inflamación/metabolismo , Degeneración del Disco Intervertebral , Lumican/genética , Lumican/metabolismo , MAP Quinasa Quinasa Quinasa 5/metabolismo , Modelos Biológicos , Núcleo Pulposo/citología , Transducción de Señal/genética , Factor de Necrosis Tumoral alfa/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
INTRODUCTION: Aortic dissection (AD) is a life-threatening emergency, and lumican (LUM) is a potential Biomarker for AD diagnosis. We investigated LUM expression patterns in patients with AD and explored the molecular functions of Lum in AD mice model. METHODS: LUM expression patterns were analyzed using aortic tissues of AD patients, and serum soluble LUM (s-LUM) levels were compared between patients with acute AD (AAD) and chronic AD (CAD). Lum-knockout (Lum-/-) mice were challenged with ß-aminopropionitrile (BAPN) and angiotensin II (Ang II) to induce AD. The survival rate, AD incidence, and aortic aneurysm (AA) in these mice were compared with those in BAPN-Ang II-challenged wildtype (WT) mice. Tgf-ß/Smad2, Mmps, Lum, and Nox expression patterns were examined. RESULTS: LUM expression was detected in the intima and media of the ascending aorta in patients with AAD. Serum s-LUM levels were significantly higher in patients with AAD than CAD. Furthermore, AD-associated mortality and thoracic aortic rupture incidence were significantly higher in the Lum-/- AD mice than in the WT AD mice. However, no significant pathologic changes in AA were observed in the Lum-/- AD mice compared with the WT AD mice. The BAPN-Ang II-challenged WT and Lum-/- AD mice had higher Tgf-ß, p-Smad2, Mmp2, Mmp9, and Nox4 levels than those of non-AD mice. We also found that Lum expression was significantly higher in the BAPN-Ang II-challenged WT in comparison to the unchallenged WT mice. CONCLUSION: LUM expression was altered in patients with AD display increased s-LUM in blood, and Lum-/- mice exhibited augmented AD pathogenesis. These findings support the notion that LUM is a biomarker signifying the pathogenesis of injured aorta seen in AAD. The presence of LUM is essential for maintenance of connective tissue integrity. Future studies should elucidate the mechanisms underlying LUM association in aortic changes.
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Disección Aórtica/patología , Lumican/sangre , Enfermedad Aguda , Aminopropionitrilo/farmacología , Disección Aórtica/metabolismo , Disección Aórtica/mortalidad , Angiotensina II/farmacología , Animales , Aorta/metabolismo , Aorta/patología , Rotura de la Aorta/epidemiología , Rotura de la Aorta/patología , Biomarcadores/sangre , Enfermedad Crónica , Modelos Animales de Enfermedad , Humanos , Incidencia , Estimación de Kaplan-Meier , Lumican/deficiencia , Lumican/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína Smad2/genética , Proteína Smad2/metabolismo , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Multiple pieces of evidence illustrate that impaired trophoblast function results in preeclampsia (PE), and migration/invasion of human trophoblast cells is stringently regulated by extracellular matrix (ECM) components. Many studies have indicated abnormal expressions of placental ECM components are associated with preeclampsia. However, the change and influence of lumican, a vital member of extracellular matrix (ECM) molecules, on trophoblast cells during preeclampsia remain unclear. This study examines the possibility that the roles of lumican in trophoblast cells contribute to PE. To address this issue, the expression of lumican in human placental tissues was observed using immunohistochemistry, fluorescence quantitative PCR, and Western blot technology. After the HTR-8/SVneo cell line was transfected with pcDNA3.1-human lumican, pGPU6-human lumican shRNA, and their negative controls, the impact of lumican on the HTR-8/SVneo cell line was investigated. Lumican was expressed in human placental tissues. Compared with the control group, its expression was significantly lower in PE placentas. Lumican downregulation inhibited cell proliferation significantly and reduced Bcl-2 expression, but increased P53 expression. These results indicate that the downregulation of placental lumican may drive PE development via promoting the downregulation of Bcl-2 expression and upregulation of P53.
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Progresión de la Enfermedad , Regulación hacia Abajo/fisiología , Lumican/biosíntesis , Placenta/metabolismo , Preeclampsia/metabolismo , Adulto , Línea Celular Transformada , Femenino , Humanos , Recién Nacido , Lumican/genética , Placenta/patología , Preeclampsia/genética , Preeclampsia/patología , EmbarazoRESUMEN
Purpose: To explore the effect of mechanical compression (similar to that induced by eye rubbing) on cell morphology, proliferation, apoptosis, and extracellular matrix synthesis and degradation in human corneal fibroblasts.Materials and methods: Human corneal fibroblasts were isolated from corneal lenticule tissue of 10 patients after small incision lenticule extraction surgery. A system was established to mechanically compress corneal fibroblasts with pressure ranging from 0 to 524 Pa. Morphological changes, cell proliferation, apoptosis, and corneal matrix synthesis and degradation were examined using microscopy imaging, bromodeoxyuridine staining, flow cytometry, and qPCR analysis in human corneal fibroblasts after mechanical compression.Results: Human corneal fibroblasts showed short and thick cytoplasmic extensions, as well as a relatively low aspect ratio, suggesting significant morphological alterations caused by high levels of compressive stress. Mechanical compression inhibited cell proliferation and promoted cell apoptosis. Furthermore, compressive stress led to significant elevation in the expression of genes related to extracellular matrix degradation (matrix metalloproteinases MMP1 and MMP9) as early as 6 h after compression and moderate changes in the expression of tissue inhibitors of metalloproteinases. In addition, the mRNA expression levels of COL1A1, lumican, and vimentin were reduced 24 and 48 h after mechanical compression.Conclusions: Mechanical compression alters cell morphology, inhibits proliferation, induces apoptosis, upregulates genes related to extracellular matrix degradation, and downregulates corneal structural genes in human corneal fibroblasts. This study provides evidence that compressive stress significantly influences corneal keratocytes. Our findings suggest that this mechanical effect may be related to keratoconus associated with chronic eye rubbing.
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Queratocitos de la Córnea/metabolismo , Queratocitos de la Córnea/patología , Estrés Mecánico , Adulto , Apoptosis , Proliferación Celular/fisiología , Células Cultivadas , Cadena alfa 1 del Colágeno Tipo I/genética , Proteínas de la Matriz Extracelular/metabolismo , Femenino , Citometría de Flujo , Humanos , Lumican/genética , Masculino , Metaloproteinasa 1 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Fenómenos Mecánicos , Reacción en Cadena de la Polimerasa , ARN Mensajero/genética , Donantes de Tejidos , Vimentina/genética , Adulto JovenRESUMEN
INTRODUCTION: Diabetic nephropathy (DN) remains a major cause of end-stage renal disease. The development of novel biomarkers and early diagnosis of DN are of great clinical importance. The goal of this study was to identify hub genes with diagnostic potential for DN by weighted gene co-expression network analysis (WGCNA). METHODS: Gene Expression Omnibus database was searched for microarray data including distinct types of CKD. Gene co-expression network was constructed, and modules specific for DN were identified by WGCNA. Gene ontology (GO) analysis was performed, and the hub genes were screened out within the selected gene modules. In addition, cross-validation was performed in an independent dataset and in samples of renal biopsies with DN and other types of glomerular diseases. RESULTS: Dataset GSE99339 was selected, and a total of 179 microdissected glomeruli samples were analyzed, including DN, normal control, and 7 groups of other glomerular diseases. Twenty-three modules of the total 10,947 genes were grouped by WGCNA, and a module was specifically correlated with DN (r = 0.54, p = 9e-15). GO analysis showed that module genes were mainly enriched in the accumulation of extracellular matrix (ECM). LUM, ELN, FBLN1, MMP2, FBLN5, and FMOD were identified as hub genes. Cross verification showed LUM and FMOD were higher in the DN group and were negatively correlated with estimated glomerular filtration rate (eGFR). In renal biopsies, expression levels of LUM and FMOD were higher in DN than IgA nephropathy, membranous nephropathy, and normal controls. CONCLUSION: By using WGCNA approach, we identified LUM and FMOD related to ECM accumulation and were specific for DN. These 2 genes may represent potential candidate diagnostic biomarkers of DN.
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Nefropatías Diabéticas/genética , Matriz Extracelular/genética , Fibromodulina/genética , Lumican/genética , Nefropatías Diabéticas/patología , Matriz Extracelular/patología , Fibromodulina/análisis , Perfilación de la Expresión Génica , Ontología de Genes , Redes Reguladoras de Genes , Humanos , Lumican/análisisRESUMEN
BACKGROUND: We developed a preclinical protocol for the screening of candidate drugs able to control myopia and prevent its progression. The protocol uses zebrafish, C57BL/6 mice, and golden Syrian hamster models of myopia. METHODS: A morpholino (MO) targeting the zebrafish lumican gene (zlum) was injected into single-cell zebrafish embryos, causing excessive expansion of the sclera. A library of 640 compounds with 2 matrix metalloproteinase (MMP) inhibitors (marimastat and batimastat), which have the potential to modulate scleral remodelling, was screened to identify candidates for mitigating scleral diameter expansion in zlum-MO-injected embryos. The myopia-prevention ability of compounds discovered to have superior potency to inhibit scleral expansion was validated over 4 weeks in 4-week-old C57BL/6 mice and 3-week-old golden Syrian hamsters with form-deprivation myopia (FDM). Changes in the refractive error and axial length were investigated. Scleral thickness, morphology of collagen fibrils in the posterior sclera, messenger RNA (mRNA) expressions, and protein levels of transforming growth factor-ß2 (TGF-ß2), tissue inhibitor of metalloproteinase-2 (TIMP-2), MMP-2, MMP-7, MMP-9, and collagen, type I, alpha 1 (collagen Iα1) were investigated in C57BL/6 mice, and MMP-2, MMP-9, and MMP activity assays were conducted in these mice. FINDINGS: In the zebrafish experiment, atropine, marimastat, batimastat, doxycycline, and minocycline were the drugs that most effectively reduced expansion of scleral equatorial diameter. After 28-day treatment in diffuser-wearing mice and 21-day treatment in lid-sutured hamsters, myopic shift and axial elongation were significantly mitigated by eye drops containing 1% atropine, 50 µM marimastat, 5 µM batimastat, or 200 µM doxycycline. MMP-2 mRNA expression in mouse sclera was lower after treatment with atropine, marimastat, batimastat, or doxycycline. The protein levels and activity of MMP-2 and MMP-7 were significantly reduced after treatment with atropine, marimastat, batimastat, doxycycline, and minocycline. Furthermore, scleral thickness and collagen fibril diameter were not lower after treatment with atropine, marimastat, batimastat, or doxycycline than those of occluded eyes. INTERPRETATION: Stepwise drug screening in a range of models from zlum-MO-injected zebrafish to rodent FDM models identified effective compounds for preclinical myopia control or prevention. On the basis of the 640 compounds that were screened, MMP inhibitors may offer alternatives for clinical trials. FUNDING: This research was supported by grants from Taiwan's Ministry of Science and Technology and Ministry of Health and Welfare.
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Inhibidores de la Metaloproteinasa de la Matriz/uso terapéutico , Miopía/tratamiento farmacológico , Animales , Atropina/uso terapéutico , Cricetinae , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Embrión no Mamífero/metabolismo , Ácidos Hidroxámicos/uso terapéutico , Lumican/antagonistas & inhibidores , Lumican/genética , Lumican/metabolismo , Metaloproteinasa 2 de la Matriz/química , Metaloproteinasa 2 de la Matriz/genética , Metaloproteinasa 2 de la Matriz/metabolismo , Ratones , Ratones Endogámicos C57BL , Morfolinos/metabolismo , Fenilalanina/análogos & derivados , Fenilalanina/uso terapéutico , Esclerótica/metabolismo , Tiofenos/uso terapéutico , Inhibidor Tisular de Metaloproteinasa-2/genética , Inhibidor Tisular de Metaloproteinasa-2/metabolismo , Pez Cebra/metabolismo , Proteínas de Pez Cebra/antagonistas & inhibidores , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
Lumican (LUM), a small leucine-rich proteoglycan, is a component of the extracellular matrix. Abnormal LUM expression is potentially associated with cancer progression. In the present study, we confirmed high LUM mRNA expression in colorectal adenocarcinoma (COAD) through the UALCAN database. The Kaplan-Meier method, univariate, and multivariate COX analysis showed that high LUM expression is an independent determinant of poor prognosis in COAD. A COX regression model was constructed based on clinical information and LUM expression. The receiver operating characteristic (ROC) curve indicated that this model was highly accurate in monitoring COAD prognosis. The co-expression network of LUM was determined by LinkedOmics, which showed that LUM expression was closely related to immune escape and the miR200 family. Furthermore, we studied the co-expression network of LUM and found that LUM could promote tumor metastasis and invasion. The Tumor Immune Estimation Resource website showed that LUM was closely related to immune infiltration and correlated with regulatory T cells, tumour-associated macrophages, and dendritic cells. We found that LUM cultivated cancer progression by targeting the miR200 family to promote epithelial-to-mesenchymal transition. These findings suggest that LUM is a potential target for inhibiting immune escape and carcinogenic pathways.
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Adenocarcinoma/genética , Neoplasias Colorrectales/genética , Lumican/genética , Escape del Tumor/genética , Adenocarcinoma/inmunología , Adenocarcinoma/patología , Neoplasias Colorrectales/inmunología , Neoplasias Colorrectales/patología , Bases de Datos Genéticas , Humanos , Lumican/inmunología , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/patología , Pronóstico , Modelos de Riesgos Proporcionales , ARN Mensajero/metabolismo , Curva ROC , Escape del Tumor/inmunologíaRESUMEN
Keloids are characterized by fibroblast activation and altered architecture of extracellular matrix (ECM). Excessive deposition of ECM molecules and irregular organization of collagen fibers have been observed in keloids. However, the ultrastructural alteration of collagen has not been fully investigated. In this study, the differences in tissue structure, collagen ultrastructure, matrix components, mechanical properties and collagen assembling molecules between keloids and their extra-lesional skins (ELSs) were explored using histology, transmission electron microscope (TEM), qPCR, Western blot, immunohistochemistry and bioinformatics. Histological evaluation showed thinner fibers in keloids with increased contents of collagen III and proteoglycans, which were supported by TEM findings of thinner collagen fibrils and less developed D-band periodicity in keloids than in ELSs (p < 0.05). In addition, total collagen and water contents were significantly increased (p < 0.05) along with richer proteoglycan production in keloids vs ELSs, which also led to increased stiffness and decreased maximal load in keloids compared with ELSs. Mechanism study showed that multiple molecules related to matrix assembly were significantly upregulated in keloids (p < 0.05). In particular, lumican and collagen V showed high degrees in co-expression analysis and their upregulation levels were revealed from microarray data, which were also verified in keloids at both gene and protein levels (p < 0.05). Nevertheless, siRNA knockdown of lumican failed to affect in vitro collagen assembly, but caused upregulated collagen V expression along with the upregulation of focal adhesion kinase, TGF-ß1, TGF-ß3 and PDGF, among which some are known for capable of enhancing collagen V expression. In conclusion, this study demonstrates impaired collagen assembly along with enhanced expression of lumican and collagen V, both are known for interfering with collagen fibril assembly.
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Colágeno Tipo V/genética , Colágeno Tipo V/metabolismo , Regulación de la Expresión Génica , Queloide/genética , Queloide/metabolismo , Lumican/genética , Adulto , Colágeno Tipo V/química , Femenino , Humanos , Masculino , Persona de Mediana Edad , Adulto JovenRESUMEN
BACKGROUND/AIM: Aneurysmal bone cyst is a benign bone lesion with a strong tendency to recur. The rearrangement of chromosome band 17p13/USP6 gene is now considered a characteristic genetic feature of aneurysmal bone cyst, with t(16;17)(q22;p13)/CDH11-USP6 as the most frequent chromosomal aberration/fusion gene. We report a novel variant translocation leading to a new fusion gene in an aneurysmal bone cyst. MATERIALS AND METHODS: Genetic analyses were performed on an aneurysmal bone cyst found in the tibia of a child. RESULTS: G-banding chromosome analysis yielded the karyotype 46,XX,t(12;17)(q21;p13)[5]/46,XX[2]. FISH analysis with a USP6 break-apart probe showed rearrangement of USP6. RNA sequencing detected LUM-USP6 and USP6-LUM fusion transcripts which were subsequently verified by RT-PCR/Sanger sequencing. The two genes exchanged 5'- non-coding exons. Thus, promoter swapping between USP6 and LUM had taken place. CONCLUSION: We report a novel t(12;17)(q21;p13) chromosome translocation which gave rise to a LUM-USP6 fusion in an aneurysmal bone cyst.
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Quistes Óseos Aneurismáticos/genética , Lumican/genética , Translocación Genética , Ubiquitina Tiolesterasa/genética , Quistes Óseos Aneurismáticos/diagnóstico , Quistes Óseos Aneurismáticos/patología , Niño , Cromosomas Humanos Par 16/genética , Cromosomas Humanos Par 17/genética , Femenino , Fusión Génica , Humanos , Hibridación Fluorescente in Situ , Cariotipificación , Regiones Promotoras Genéticas/genética , Análisis de Secuencia de ARNRESUMEN
Chondrosarcoma is a malignant bone tumor characterized by the production of a modified cartilagetype extracellular matrix (ECM). In the present study, the expression levels of the small leucinerich proteoglycans (SLRPs), decorin, biglycan and lumican, were examined in the HTB94 human chondrosarcoma cell line. HTB94 cells were found to express and secrete the 3 SLRP members. RTqPCR and western blot analysis demonstrated that lumican was the most abundantly secreted SLRP, whereas decorin and biglycan expression levels were low. The utilization of short interfering RNA specific for the decorin, biglycan, and lumican genes resulted in the efficient downregulation of the respective mRNA levels (P≤0.001). The growth of the HTB94 cells was stimulated by lumican (P≤0.001), whereas their migration and adhesion were not affected (P=NS). By contrast, these cellular functions were not sensitive to a decrease in low endogenous levels of decorin and biglycan. Lumicandeficiency significantly inhibited both basal and insulinlike growth factor I (IGFI)induced HTB94 cell growth (P≤0.001 andP≤0.01, respectively). These effects were executed through the insulinlike growth factor I receptor (IGFIR), whose activation was markedly attenuated (P≤0.01) in lumicandeficient HTB94 cells. The downregulation of lumican induced the substantial inhibition of extracellular regulated kinase (ERK1/2) activation (P≤ 0.01), indicating that ERK1/2 is a necessary component of lumican/IGFIRmediated HTB94 cell proliferation. Moreover, the lumicandeficient cells exhibit increased mRNA levels of p53 (P≤0.05), suggesting that lumican facilitates HTB94 cell growth through an IGFIR/ERK1/2/p53 signaling cascade. On the whole, the findings of the present study demonstrate that endogenous lumican is a novel regulator of HTB94 cell growth.
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Neoplasias Óseas/patología , Condrosarcoma/patología , Lumican/metabolismo , Línea Celular Tumoral , Proliferación Celular , Humanos , Factor I del Crecimiento Similar a la Insulina/metabolismo , Lumican/genética , Sistema de Señalización de MAP Quinasas , ARN Interferente Pequeño/metabolismo , Receptor IGF Tipo 1/metabolismoRESUMEN
INTRODUCTION: The heart undergoes myocardial remodeling during progression to heart failure following pressure overload. Myocardial remodeling is associated with structural and functional changes in cardiac myocytes, fibroblasts, and the extracellular matrix (ECM) and is accompanied by inflammation. Cardiac fibrosis, the accumulation of ECM molecules including collagens and collagen cross-linking, contributes both to impaired systolic and diastolic function. Insufficient mechanistic insight into what regulates cardiac fibrosis during pathological conditions has hampered therapeutic so-lutions. Lumican (LUM) is an ECM-secreted proteoglycan known to regulate collagen fibrillogenesis. Its expression in the heart is increased in clinical and experimental heart failure. Furthermore, LUM is important for survival and cardiac remodeling following pressure overload. We have recently reported that total lack of LUM increased mortality and left ventricular dilatation, and reduced collagen expression and cross-linking in LUM knockout mice after aortic banding (AB). Here, we examined the effect of LUM on myocardial remodeling and function following pressure overload in a less extreme mouse model, where cardiac LUM level was reduced to 50% (i.e., moderate loss of LUM). METHODS AND RESULTS: mRNA and protein levels of LUM were reduced to 50% in heterozygous LUM (LUM+/-) hearts compared to wild-type (WT) controls. LUM+/- mice were subjected to AB. There was no difference in survival between LUM+/- and WT mice post-AB. Echocardiography revealed no striking differences in cardiac geometry between LUM+/- and WT mice 2, 4, and 6 weeks post-AB, although markers of diastolic dysfunction indicated better function in LUM+/- mice. LUM+/- hearts revealed reduced cardiac fibrosis assessed by histology. In accordance, the expression of collagen I and III, the main fibrillar collagens in the heart, and other ECM molecules central to fibrosis, i.e. including periostin and fibronectin, was reduced in the hearts of LUM+/- compared to WT 6 weeks post-AB. We found no differences in collagen cross-linking between LUM+/- and WT mice post-AB, as assessed by histology and qPCR. CONCLUSIONS: Moderate lack of LUM attenuated cardiac fibrosis and improved diastolic dysfunction following pressure overload in mice, adding to the growing body of evidence suggesting that LUM is a central profibrotic molecule in the heart that could serve as a potential therapeutic target.
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Insuficiencia Cardíaca/metabolismo , Ventrículos Cardíacos/metabolismo , Hipertrofia Ventricular Izquierda/metabolismo , Lumican/fisiología , Miofibroblastos/metabolismo , Animales , Colágeno/metabolismo , Modelos Animales de Enfermedad , Ecocardiografía , Matriz Extracelular/metabolismo , Ventrículos Cardíacos/patología , Lumican/genética , Masculino , Ratones , Ratones Noqueados , Miofibroblastos/patología , Remodelación VentricularRESUMEN
Lumican is overexpressed in lung cancer cells and has been implicated in the pathogenesis of tumorigenesis and regulation of cancer cell invasion. Lumican is robustly associated with the binding of p120-catenin protein to modulate cell metastasis. However, its role in cancer cell proliferation is still unclear. This study investigated the effect of lumican on the cell division including mitosis and cytokinesis in non-small lung cancer cells. We found that the downregulation of lumican prolonged the doubling time of cells and retarded the cell growth in H460 and A549 cells. Along with tubulin, lumican localized to the mitotic spindle and centrosome during the metaphase-anaphase stage. The cell cycle was retained in the G2/M phase after the downregulation of lumican. Interestingly, lumican was found to play important roles in central spindle and midbody formation during cytokinesis. Lumican interacted with the midbody-associated proteins such as MKLP1, Aurora B, and ECT2. Notably, the downregulation of lumican decreased the level of MKLP1 accompanied by the retention of midbody-residual that resulted in multi-nucleated cells. Downregulation of lumican promoted the chromosome missegregation and the increment of the bi-/multinucleated cells. The results of this study indicated that lumican associated with tubulin is crucial for spindle fiber formation and midbody assembly in cell division. Downregulation of lumican displayed the defects in mitotic spindle assembly/dynamics and improper kinetochore-microtubules attachment that led to increase aneuploidy. This emerging property of lumican is suggested to tightly control chromosome segregation during cell division in lung cancer cells.Abbreviations: ESCRT: endosomal sorting complex required for transport; PRC1: protein regulator of cytokinesis 1; Nci: negative control siRNA; Lumi: lumican siRNAs; MKLP1: mitotic kinesin-like protein 1; H460LD and A549LD: H460 and A549 cell lines with less expressed lumican.
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Aneuploidia , Regulación hacia Abajo/genética , Lumican/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Mitosis/genética , Línea Celular Tumoral , Proliferación Celular , Citocinesis/genética , Humanos , Lumican/metabolismo , Microtúbulos/metabolismo , Proteínas Proto-Oncogénicas/metabolismoRESUMEN
A great hallmark of breast cancer is the absence or presence of estrogen receptors ERα and ERß, with a dominant role in cell proliferation, differentiation and cancer progression. Both receptors are related with Epithelial-to-Mesenchymal Transition (EMT) since there is a relation between ERs and extracellular matrix (ECM) macromolecules expression, and therefore, cell-cell and cell-ECM interactions. The endocrine resistance of ERα endows epithelial cells with increased aggressiveness and induces cell proliferation, resulting into a mesenchymal phenotype and an EMT status. ERα signaling may affect the transcriptional factors which govern EMT. Knockdown or silencing of ERα and ERß in MCF-7 and MDA-MB-231 breast cancer cells respectively, provoked pivotal changes in phenotype, cellular functions, mRNA and protein levels of EMT markers, and consequently the EMT status. Mesenchymal cells owe their migratory and invasive properties to invadopodia, while in epithelial cells, lamellipodia and filopodia are mostly observed. Invadopodia, are actin-rich protrusions of plasma membrane, promoting proteolytic degradation of ECM and tumor invasion. Cortactin and MMP-14 govern the formation and principal functions of invadopodia. In vitro experiments proved that lumican inhibits cortactin and MMP-14 expression, alters the formation of lamellipodia and transforms mesenchymal cells into epithelial-like. Conclusively, lumican may inhibit or even reverse the several metastatic features that EMT endows in breast cancer cells. Therefore, a lumican-based anti-cancer therapy which will pharmacologically target and inhibit EMT might be interesting to be developed.
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Biomarcadores de Tumor , Neoplasias de la Mama/etiología , Neoplasias de la Mama/metabolismo , Transición Epitelial-Mesenquimal , Podosomas/metabolismo , Animales , Neoplasias de la Mama/patología , Neoplasias de la Mama/terapia , Transición Epitelial-Mesenquimal/genética , Matriz Extracelular/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Lumican/genética , Lumican/metabolismo , Terapia Molecular Dirigida , Transducción de SeñalRESUMEN
The transcription factor FOXO3 is associated with poor outcome in high-stage neuroblastoma (NB), as it facilitates chemoprotection and tumor angiogenesis. In other tumor entities, FOXO3 stimulates metastasis formation, one of the biggest challenges in the treatment of aggressive NB. However, the impact of FOXO3 on the metastatic potential of neuronal tumor cells remains largely unknown. In the present study, we uncover the small leucine-rich proteoglycan family member lumican (LUM) as a FOXO3-regulated gene that stimulates cellular migration in NB. By a drug-library screen we identified the small molecular weight compound repaglinide (RPG) as a putative FOXO3 inhibitor. Here, we verify that RPG binds to the FOXO3-DNA-binding-domain (DBD) and thereby silences the transcriptional activity of FOXO3. Consistent with the concept that the FOXO3/LUM axis enhances the migratory capacity of aggressive NB cells, we demonstrate that stable knockdown of LUM abrogates the FOXO3-mediated increase in cellular migration. Importantly, FOXO3 inhibition by RPG represses the binding of FOXO3 to the LUM promoter, inhibits FOXO3-mediated LUM RNA and protein expression, and efficiently abrogates FOXO3-triggered cellular "wound healing" as well as spheroid-based 3D-migration. Thus, silencing the FOXO3/LUM axis by the FDA-approved compound RPG represents a promising strategy for novel therapeutic interventions in NB and other FOXO3-dependent tumors.