RESUMO
Lung cancer is a leading cause of cancer-related mortality globally, with a dismal 5-year survival rate, particularly for Lung Adenocarcinoma (LUAD). Mechanical changes within the tumor microenvironment, such as extracellular matrix (ECM) remodeling and fibroblast activity, play pivotal roles in cancer progression and metastasis. However, the specific impact of the basement membrane (BM) on the mechanical characteristics of LUAD remains unclear. This study aims to identify BM genes influencing internal mechanical stress in tumors, elucidating their effects on LUAD metastasis and therapy resistance, and exploring strategies to counteract these effects. Using Matrigel overlay and Transwell assays, we found that mechanical stress, mimicked by matrix application, augmented LUAD cell migration and invasion, correlating with ECM alterations and activation of the epithelial-mesenchymal transition (EMT) pathway. Employing machine learning, we developed the SVM_Score model based on relevant BM genes, which accurately predicted LUAD patient prognosis and EMT propensity across multiple datasets. Lower SVM_Scores were associated with worse survival outcomes, elevated cancer-related pathways, increased Tumor Mutation Burden, and higher internal mechanical stress in LUAD tissues. Notably, the SVM_Score was closely linked to COL5A1 expression in myofibroblasts, a key marker of mechanical stress. High COL5A1 expression from myofibroblasts promoted tumor invasiveness and EMT pathway activation in LUAD cells. Additionally, treatment with Sorafenib, which targets COL5A1 secretion, attenuated the tumor-promoting effects of myofibroblast-derived COL5A1, inhibiting LUAD cell proliferation, migration, and enhancing chemosensitivity. In conclusion, this study elucidates the complex interplay between mechanical stress, ECM alterations, and LUAD progression. The SVM_Score emerges as a robust prognostic tool reflecting tumor mechanical characteristics, while Sorafenib intervention targeting COL5A1 secretion presents a promising therapeutic strategy to mitigate LUAD aggressiveness. These findings deepen our understanding of the biomechanical aspects of LUAD and offer insights for future research and clinical applications.
Assuntos
Adenocarcinoma de Pulmão , Colágeno Tipo V , Transição Epitelial-Mesenquimal , Neoplasias Pulmonares , Miofibroblastos , Estresse Mecânico , Humanos , Adenocarcinoma de Pulmão/patologia , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/metabolismo , Adenocarcinoma de Pulmão/tratamento farmacológico , Miofibroblastos/metabolismo , Miofibroblastos/efeitos dos fármacos , Miofibroblastos/patologia , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/tratamento farmacológico , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Colágeno Tipo V/metabolismo , Colágeno Tipo V/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Linhagem Celular Tumoral , Animais , Movimento Celular/efeitos dos fármacos , Metástase Neoplásica , Camundongos , Microambiente Tumoral , Sorafenibe/farmacologia , Sorafenibe/uso terapêutico , Matriz Extracelular/metabolismoRESUMO
Triple-negative breast cancer (TNBC) is an exceptionally aggressive subtype of breast cancer. Despite the recognized interplay between tumors and tumor-associated macrophages in fostering drug resistance and disease progression, the precise mechanisms leading these interactions remain elusive. Our study revealed that the upregulation of collagen type V alpha 1 (COL5A1) in TNBC tissues, particularly in chemoresistant samples, was closely linked to an unfavorable prognosis. Functional assays unequivocally demonstrated that COL5A1 played a pivotal role in fueling cancer growth, metastasis, and resistance to doxorubicin, both in vitro and in vivo. Furthermore, we found that the cytokine IL-6, produced by COL5A1-overexpressing TNBC cells actively promoted M2 macrophage polarization. In turn, TGFß from M2 macrophages drived TNBC doxorubicin resistance through the TGFß/Smad3/COL5A1 signaling pathway, establishing a feedback loop between TNBC cells and macrophages. Mechanistically, COL5A1 interacted with TGM2, inhibiting its K48-linked ubiquitination-mediated degradation, thereby enhancing chemoresistance and increasing IL-6 secretion. In summary, our findings underscored the significant contribution of COL5A1 upregulation to TNBC progression and chemoresistance, highlighting its potential as a diagnostic and therapeutic biomarker for TNBC.
Assuntos
Colágeno Tipo V , Progressão da Doença , Resistencia a Medicamentos Antineoplásicos , Neoplasias de Mama Triplo Negativas , Humanos , Neoplasias de Mama Triplo Negativas/patologia , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/genética , Feminino , Colágeno Tipo V/metabolismo , Colágeno Tipo V/genética , Camundongos , Animais , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Macrófagos/metabolismo , Macrófagos/patologia , Interleucina-6/metabolismo , Interleucina-6/genética , Doxorrubicina/farmacologia , Macrófagos Associados a Tumor/metabolismo , Macrófagos Associados a Tumor/patologia , Transdução de Sinais , Proteína 2 Glutamina gama-Glutamiltransferase/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteínas de Ligação ao GTP/metabolismo , Proteínas de Ligação ao GTP/genéticaRESUMO
Gastric cancer is a type of digestive tract cancer with a high morbidity and mortality, which leads to a major health burden worldwide. More research into the functions of the immune system will improve therapy and survival in gastric cancer patients. We attempted to identify potential biomarkers or targets in gastric cancer via bioinformatical analysis approaches. Three gene expression profile datasets (GSE79973, GSE103236, and GSE118916) of gastric tissue samples were obtained from the Gene Expression Omnibus database. There were 65 overlapping differentially expressed genes (DEGs) identified from three microarrays. Gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway were carried out for the key functions and pathways enriched in the DEGs. Then, ten hub genes were identified by protein-protein interaction network. In addition, we observed that collagen type V alpha 2 (COL5A2) was linked to gastric cancer prognosis as well as M2 macrophage infiltration. Furthermore, COL5A2 enhanced gastric cancer cell proliferation through the PI3K-AKT signaling pathway and polarized M2 macrophage cells. Therefore, in this study, we found that COL5A2 was associated with the development of gastric cancer which might function as a potential therapeutic target for the disease.
Assuntos
Colágeno Tipo V , Perfilação da Expressão Gênica , Neoplasias Gástricas , Humanos , Biomarcadores Tumorais/genética , Colágeno Tipo V/genética , Colágeno Tipo V/metabolismo , Biologia Computacional , Regulação Neoplásica da Expressão Gênica , Macrófagos , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Neoplasias Gástricas/genéticaRESUMO
BACKGROUND AND AIMS: Liver-associated complications still frequently lead to mortality in people with HIV (PWH), even though combined antiretroviral treatment (cART) has significantly improved overall survival. The quantification of circulating collagen fragments released during collagen formation and degradation correlate with the turnover of extracellular matrix (ECM) in liver disease. Here, we analysed the levels of ECM turnover markers PC3X, PRO-C5, and PRO-C6 in PWH and correlated these with hepatic fibrosis and steatosis. METHODS: This monocentre, retrospective study included 141 PWH. Liver stiffness and liver fat content were determined using transient elastography (Fibroscan) with integrated CAP function. Serum levels of formation of cross-linked type III collagen (PC3X), formation of type V collagen (PRO-C5) and formation type VI collagen (PRO-C6), also known as the hormone endotrophin, were measured with ELISA. RESULTS: Twenty-five (17.7%) of 141 PWH had clinical significant fibrosis with liver stiffness ≥ 7.1 kPa, and 62 PWH (44.0%) had steatosis with a CAP value > 238 dB/m. Study participants with fibrosis were older (p = 0.004) and had higher levels of AST (p = 0.037) and lower number of thrombocytes compared to individuals without fibrosis (p = 0.0001). PC3X and PRO-C6 were markedly elevated in PWH with fibrosis. Multivariable cox regression analysis confirmed PC3X as independently associated with hepatic fibrosis. PRO-C5 was significantly elevated in participants with presence of hepatic steatosis. CONCLUSION: Serological levels of cross-linked type III collagen formation and endotrophin were significantly associated with liver fibrosis in PWH receiving cART and thus may be suitable as a non-invasive evaluation of liver fibrosis in HIV disease.
Assuntos
Colágeno Tipo III , Colágeno Tipo VI , Colágeno Tipo V , Fígado Gorduroso , Infecções por HIV , Cirrose Hepática , Humanos , Biomarcadores/sangue , Biomarcadores/metabolismo , Colágeno Tipo III/sangue , Colágeno Tipo III/metabolismo , Colágeno Tipo VI/sangue , Colágeno Tipo VI/metabolismo , Fígado Gorduroso/sangue , Fígado Gorduroso/complicações , Fígado Gorduroso/diagnóstico por imagem , Fígado Gorduroso/metabolismo , Infecções por HIV/sangue , Infecções por HIV/complicações , Infecções por HIV/tratamento farmacológico , Infecções por HIV/metabolismo , Fígado/diagnóstico por imagem , Fígado/metabolismo , Cirrose Hepática/sangue , Cirrose Hepática/diagnóstico por imagem , Cirrose Hepática/etiologia , Cirrose Hepática/metabolismo , Estudos Retrospectivos , Matriz Extracelular/metabolismo , Terapia Antirretroviral de Alta Atividade , Colágeno Tipo V/sangue , Colágeno Tipo V/metabolismo , Pró-Colágeno/sangue , Pró-Colágeno/metabolismoRESUMO
Long noncoding RNAs (lncRNAs) are known to participate in the progression of several cancers, including esophageal carcinoma (EC), a common malignancy of the digestive system. Although the role of the lncRNA-miRNA-mRNA regulatory network is crucial for the growth and progression of EC, the regulation of lncRNA BBOX1-AS1 (BBOX1 antisense RNA1) remains unclear. We performed reverse transcription-quantitative PCR (RT-qPCR) and western blotting to evaluate miR-361-3p, collagen type V alpha 1 chain (COL5A1), and BBOX1-AS1 expression levels in EC cells and tissues. The colony formation assay (CFA) and Cell Counting Kit-8 (CCK-8) were employed to identify EC cell proliferation, while western blotting was used to examine EC cell apoptosis and Bax and Bcl-2 expression levels. The effect of BBOX1-AS1 on EC proliferation was determined using an in vivo carcinogenesis assay. Correlation between COL5A1, BBOX1-AS1, and miR-361-3p was examined using the luciferase reporter system and RNA immunoprecipitation assay (RIP). Herein, we observed that BBOX1-AS1 expression levels were upregulated in EC cells and tissues. BBOX1-AS1 knockdown inhibited EC cell proliferation and conferred a pro-apoptotic effect. These results indicated a positive interaction between BBOX1-AS1 and miR-361-3p in EC and a negative association with miR-361-3p. COL5A1 was recognized as a downstream miR-361-3p target and was inversely related to miR-361-3p in EC. Therefore, BBOX1-AS1 expression suppressed cell apoptosis and promoted cell proliferation via the downregulation of miR-361-3p and upregulation of COL5A1 expression. Overall, BBOX1-AS1 facilitates EC progression via the miR-361-3p or COL5A1 axis, indicating that BBOX1-AS1 might be a potential therapeutic target for EC therapy.
Assuntos
Carcinoma , MicroRNAs , RNA Longo não Codificante , Humanos , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Linhagem Celular Tumoral , Movimento Celular/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Colágeno/metabolismo , Carcinoma/genética , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica , Colágeno Tipo V/genética , Colágeno Tipo V/metabolismoRESUMO
Spermatogonial stem cells (SSCs) are the basis of spermatogenesis. Systematically exploring the critical factors associated with the formation of SSCs will provide new insight to improve the formation efficiency, and their practical application. Here we explore the regulatory mechanism of the ECM-receptor interaction signaling pathway and related genes during differentiation of SSCs in chicken. Firstly, the positive cell rate of SSCs protein marker was detected by immunofluorescence and flow cytometry and qRT-PCR was used to identify, the expression of related marker genes after 10 days of RA-induction. Secondly, the ESCs on 0d/ 4d /10d after RA- induction/self-differentiation were collected, and the total RNA was then extracted from cells. Finally, high-throughput analysis methods (RNA-seq) were used to sequence the transcriptome of these cells. After PCA analysis of the RNA-seq data, Venny analysis, GO and KEGG enrichment were further used to find the key signaling pathways and genes in the RA-induction process. The results showed that on day 10 of RA-induction, grape cluster growth cells expressed integrinß1, the specific marker protein of SSCs cells, and the integrinß1 positive rate was 35.1%. Also, SSCs marker genes CVH, Integrinß1, Integrinα6 were significantly up-regulated during RA-induction. Moreover, the significantly enriched pathway, ECM-receptor interaction signaling, in current study may play a crucial role in RA-induction. Then, JASPAR was used to predict the differential gene transcription factors in the signaling pathway, finding that RA receptor was a transcription factor of COL5A1, COL5A2 and COL3A1. The qRT-PCR results showed that the expression levels of RA receptors (RXRA, RARA and RXRG) and the predicted genes (COL5A1, COL5A2 and COL3A1) were both significantly increased during RA-induction. Also, dual-luciferase reporter assay showed that RA could affect the luciferin activities of COL5A1, COL5A2 and COL3A1. These results suggest that RA plays a crucial role in the formation of chicken spermatogonial stem cells via the transcription levels of COL5A1, COL5A2 and COL3A1 to regulate the ECM-receptor interaction signaling pathway. Additionally, knockdown of COL5A1/COL5A2/COL3A1 could effectively reduce the formation efficiency of SSCs. This indicated that the interference of RA receptor binding genes in the ECM-receptor interaction signaling pathway could decrease the efficiency of RA induced SSCs formation. Therefore, this study concludes that RA promotes formation of chicken spermatogonial stem cells by regulating the ECM-receptor interaction signaling pathway.
Assuntos
Células-Tronco Germinativas Adultas/efeitos dos fármacos , Células-Tronco Germinativas Adultas/metabolismo , Receptores de Superfície Celular/metabolismo , Transdução de Sinais , Espermatogônias/efeitos dos fármacos , Espermatogônias/metabolismo , Tretinoína/farmacologia , Animais , Diferenciação Celular , Galinhas , Colágeno Tipo III/genética , Colágeno Tipo III/metabolismo , Colágeno Tipo V/genética , Colágeno Tipo V/metabolismo , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes/métodos , MasculinoRESUMO
The epidermal basement membrane deteriorates with aging. We previously reported that basement membrane reconstruction not only serves to maintain epidermal stem/progenitor cells in the epidermis, but also increases collagen fibrils in the papillary dermis. Here, we investigated the mechanism of the latter action. Collagen fibrils in the papillary dermis were increased in organotypic human skin culture treated with matrix metalloproteinase and heparinase inhibitors. The expression levels of COL5A1 and COL1A1 genes (encoding collagen type V α 1 chain and collagen type I α 1 chain, respectively) were increased in fibroblasts cultured with conditioned medium from a skin equivalent model cultured with the inhibitors and in keratinocytes cultured on laminin-511 E8 fragment-coated plates. We then examined cytokine expression, and found that the inhibitors increased the expression of PDGF-BB (platelet-derived growth factor consisting of two B subunits) in epidermis. Expression of COL5A1 and COL1A1 genes was increased in cultured fibroblasts stimulated with PDGF-BB. Further, the bifunctional inhibitor hydroxyethyl imidazolidinone (HEI) increased skin elasticity and the thickness of the papillary dermis in the skin equivalent. Taken together, our data suggests that reconstructing the basement membrane promotes secretion of PDGF-BB by epidermal keratinocytes, leading to increased collagen expression at the papillary dermis.
Assuntos
Membrana Basal/fisiologia , Epiderme/fisiologia , Colágenos Associados a Fibrilas/fisiologia , Fibroblastos/metabolismo , Fibroblastos/fisiologia , Regeneração/fisiologia , Envelhecimento da Pele/patologia , Envelhecimento da Pele/fisiologia , Membrana Basal/metabolismo , Becaplermina/genética , Becaplermina/metabolismo , Células Cultivadas , Cadeia alfa 1 do Colágeno Tipo I/genética , Cadeia alfa 1 do Colágeno Tipo I/metabolismo , Colágeno Tipo V/genética , Colágeno Tipo V/metabolismo , Células Epidérmicas/metabolismo , Epiderme/metabolismo , Epiderme/patologia , Colágenos Associados a Fibrilas/genética , Colágenos Associados a Fibrilas/metabolismo , Expressão Gênica , Humanos , Queratinócitos/metabolismo , Metaloproteinases da Matriz/farmacologia , Regeneração/genéticaRESUMO
BACKGROUND: Collagen type V alpha 1 chain (COL5A1) is a hypoxia-related gene (a collagen family protein) and participates in the formation of the extracellular matrix. Although some evidence supports a significant role for COL5A1 in the progression of several cancers, a pan-cancer analysis of COL5A1 is not currently available. Herein, we aimed to assess the prognostic value of COL5A1 in 33 human cancers and to investigate its underlying immunological function. METHODS: Through multiple bioinformatics methods, we analyzed the data from Oncomine, TCGA, CCLE, HPA, DNMIVD, and cBioPortal database to explore the potential underlying carcinogenic effect of COL5A1, including the relevance of COL5A1 to the outcome, DNA methylation, tumor microenvironment, immune cells infiltration, and drug sensitivity in 33 human cancers. The effects of COL5A1 on glioma cell proliferation, migration, and invasion were verified in cellular experiments. RESULTS: Our findings indicated that COL5A1 was expressed at high levels in 13 cancers and was negatively related to the prognosis of 11 cancers. Additionally, COL5A1 was coexpressed with genes encoding the major histocompatibility complex, immune activators, immune suppressors, chemokines, chemokine receptors, mismatch repair genes, and immune checkpoints. We also identified different roles for COL5A1 in the immunocyte infiltration in different cancers. The correlation between COL5A1 and drug sensitivity was found in several cancers. COL5A1 potentially influenced the tumor progression through immune-related pathways, negative regulation of immune system processes, chemokine signaling pathways, JAK-STAT pathways, T cell receptor pathways, lymphocyte migration, and antigen processing and presentation, among other processes. CONCLUSIONS: Based on our study, COL5A1 may be employed as a prognostic marker in different malignancies because of its impact on tumorigenesis and immune cell infiltration and have implications for cancer immune checkpoint inhibitors and chemotherapy.
Assuntos
Biomarcadores Tumorais/metabolismo , Colágeno Tipo V/metabolismo , Neoplasias/genética , Humanos , Prognóstico , Microambiente TumoralRESUMO
BACKGROUND: Keratoconus (KC) is a corneal disorder, associated with oxidative stress, hypoxia and as several times discussed, potentially with thyroid gland dysfunction. We aimed to investigate the effect of thyroxine on transforming growth factor ß1 (TGF-ß1), collagen I and V (Col I and V) expression in human corneal fibroblasts (HCFs) and human keratocytes of KC corneas, in vitro. METHODS: Primary human KC-keratocytes and normal keratocytes were isolated and cultured as corneal fibroblasts or keratocytes. The effect of 0.1 µg/ml and 1.0 µg/ml thyroxine on TGF-ß1, Col I and Col V expression was investigated by qPCR, Western blot, and ELISA. Proliferation assay was performed using BrdU ELISA to observe the 24h effect of 1.0 µg/ml thyroxine on keratocytes, in vitro. RESULTS: TGFB1 mRNA expression of normal keratocytes increased following 1.0 µg/ml thyroxine stimulation for 24 h (p = .036), without changes in protein expression. Col I protein expression of KC-HCFs increased following 1.0 µg/ml thyroxine stimulation for 24 h (p = .0003). Proliferation of normal and KC keratocytes increased following a 7-day growth period and 24 hours thyroxine administration (p = .018; p = .024). CONCLUSIONS: Thyroxine may affect the Col I protein expression in KC-HCFs, but not in KC keratocytes, in vitro. Thyroxine administration has no effect on TGF-ß1, collagen I and V expression of keratoconus keratocytes. Therefore, an increased thyroxine concentration alone seems not to be causally related to the development of keratoconus.
Assuntos
Colágeno Tipo V/metabolismo , Ceratocone , Células Cultivadas , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Córnea/metabolismo , Fibroblastos/metabolismo , Humanos , Ceratocone/tratamento farmacológico , Ceratocone/genética , Ceratocone/metabolismo , Tiroxina/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Fator de Crescimento Transformador beta1/farmacologiaRESUMO
The COL1A1 and COL5A1 variants have been associated with the risk of musculoskeletal injuries. Therefore, the main aim of the study was to investigate the association between three polymorphisms within two genes (rs1800012 in COL1A1, as well as rs12722 and rs13946 in COL5A1) and the reported, yet rarely described in the literature, injuries of the joint and muscle area in a physically active Caucasian population. Polish students (n = 114) were recruited and divided into the following two groups: students with (n = 53) and without (n = 61) injures. Genotyping was carried out using real-time PCR. The results obtained revealed a statistically significant association between rs1800012 COL1A1 and injury under an overdominant model. Specifically, when adjusted for age and sex, the GT heterozygotes had a 2.2 times higher chance of being injured compared with both homozygotes (TT and GG, 95% CI 0.59-5.07, p = 0.040). However, no significant interaction between the COL5A1 variants, either individually or in haplotype combination, and susceptibility to injury were found. In addition, the gene-gene interaction analysis did not reveal important relationships with the musculoskeletal injury status. It was demonstrated that rs1800012 COL1A1 may be positively associated with physical activity-related injuries in a Caucasian population. Harboring the specific GT genotype may be linked to a higher risk of being injured.
Assuntos
Cadeia alfa 1 do Colágeno Tipo I/metabolismo , Colágeno Tipo V/metabolismo , Predisposição Genética para Doença , Doenças Musculoesqueléticas/patologia , Polimorfismo de Nucleotídeo Único , População Branca/genética , Adulto , Estudos de Casos e Controles , Cadeia alfa 1 do Colágeno Tipo I/genética , Colágeno Tipo V/genética , Feminino , Humanos , Masculino , Doenças Musculoesqueléticas/genética , Doenças Musculoesqueléticas/metabolismo , Adulto JovemRESUMO
Diabetic keratopathy is a corneal complication of diabetes mellitus (DM). Patients with diabetic keratopathy are prone to developing corneal haze, scarring, recurrent erosions, and significant wound healing defects/delays. The purpose of this study was to determine the contractility profiles in the diabetic human corneal stromal cells and characterize their molecular signatures. Primary human corneal fibroblasts from healthy, Type 1 DM (T1DM), and Type 2 DM (T2DM) donors were cultured using an established 3D collagen gel model. We tracked, measured, and quantified the contractile footprint over 9 days and quantified the modulation of specific corneal/diabetes markers in the conditional media and cell lysates using western blot analysis. Human corneal fibroblasts (HCFs) exhibited delayed and decreased contractility compared to that from T1DMs and T2DMs. Compared to HCFs, T2DMs demonstrated an initial downregulation of collagen I (day 3), followed by a significant upregulation by day 9. Collagen V was significantly upregulated in both T1DMs and T2DMs based on basal secretion, when compared to HCFs. Cell lysates were upregulated in the myofibroblast-associated marker, α-smooth muscle actin, in T2DMs on day 9, corresponding to the significant increase in contractility rate observed at the same time point. Furthermore, our data demonstrated a significant upregulation in IGF-1 expression in T2DMs, when compared to HCFs and T1DMs, at day 9. T1DMs demonstrated significant downregulation of IGF-1 expression, when compared to HCFs. Overall, both T1DMs and T2DMs exhibited increased contractility associated with fibrotic phenotypes. These findings, and future studies, may contribute to better understanding of the pathobiology of diabetic keratopathy and ultimately the development of new therapeutic approaches.
Assuntos
Forma Celular/fisiologia , Doenças da Córnea/patologia , Substância Própria/citologia , Fibroblastos/citologia , Células Estromais/citologia , Técnicas de Cultura de Células em Três Dimensões/métodos , Células Cultivadas , Colágeno Tipo I/metabolismo , Colágeno Tipo III/metabolismo , Colágeno Tipo V/metabolismo , Doenças da Córnea/etiologia , Doenças da Córnea/metabolismo , Substância Própria/metabolismo , Diabetes Mellitus Tipo 1/complicações , Diabetes Mellitus Tipo 2/complicações , Fibroblastos/metabolismo , Humanos , Fator de Crescimento Insulin-Like I/metabolismo , Pessoa de Meia-Idade , Receptor IGF Tipo 1/metabolismo , Células Estromais/metabolismo , Fatores de TempoAssuntos
Colágeno Tipo V/metabolismo , Acetiltransferases N-Terminal/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias Gástricas/metabolismo , Acetilação , Linhagem Celular Tumoral , Colágeno Tipo V/genética , Humanos , Acetiltransferases N-Terminal/genética , Metástase Neoplásica , Proteínas de Neoplasias/genética , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologiaRESUMO
Pheochromocytoma, as a neuroendocrine tumor with the highest genetic correlation in all types of tumors, has attracted extensive attention. Von Hipper Lindau (VHL) has the highest mutation frequency among the genes associated with pheochromocytoma. However, the effect of VHL on the proteome of pheochromocytoma remains to be explored. In this study, the VHL knockdown (VHL-KD) PC12 cell model was established by RNA interference (shRNA). We compared the proteomics of VHL-KD and VHL-WT PC12 cell lines. The results showed that the expression of 434 proteins (VHL shRNA/WT > 1.3) changed significantly in VHL-KD-PC12 cells. Among the 434 kinds of proteins, 83 were involved in cell proliferation, cell cycle and cell migration, and so on. More importantly, among these proteins, we found seven novel key genes, including Connective Tissue Growth Factor (CTGF), Syndecan Binding Protein (SDCBP), Cysteine Rich Protein 61 (CYR61/CCN1), Collagen Type III Alpha 1 Chain (COL3A1), Collagen Type I Alpha 1 Chain (COL1A1), Collagen Type V Alpha 2 Chain (COL5A2), and Serpin Family E Member 1 (SERPINE1), were overexpressed and simultaneously regulated cell proliferation and migration in VHL-KD PC12 cells. Furthermore, the abnormal accumulation of HIF2α caused by VHL-KD significantly increased the expression of these seven genes during hypoxia. Moreover, cell-counting, scratch, and transwell assays demonstrated that VHL-KD could promote cell proliferation and migration, and changed cell morphology. These findings indicated that inhibition of VHL expression could promote the development of pheochromocytoma by activating the expression of cell proliferation and migration associated genes.
Assuntos
Neoplasias das Glândulas Suprarrenais/genética , Movimento Celular , Proliferação de Células , Feocromocitoma/genética , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Neoplasias das Glândulas Suprarrenais/metabolismo , Neoplasias das Glândulas Suprarrenais/fisiopatologia , Cadeia alfa 1 do Colágeno Tipo I/genética , Cadeia alfa 1 do Colágeno Tipo I/metabolismo , Colágeno Tipo III/genética , Colágeno Tipo III/metabolismo , Colágeno Tipo V/genética , Colágeno Tipo V/metabolismo , Fator de Crescimento do Tecido Conjuntivo/genética , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Proteína Rica em Cisteína 61/genética , Proteína Rica em Cisteína 61/metabolismo , Humanos , Mutação , Feocromocitoma/metabolismo , Feocromocitoma/fisiopatologia , Sinteninas/genética , Sinteninas/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismoRESUMO
BACKGROUND: Classical Ehlers-Danlos syndrome (cEDS) is a heterogeneous connective tissue disorder that mainly results from the germline mutation of COL5A1 and COL5A2. The majority of the COL5A2 mutations reported to date represent structural mutations, including missense or in-frame exon-skipping splice mutations. The only reported synonymous mutation was expected to affect on splicing of exon 29 by prediction programs which should be further confirmed. METHODS: Whole exome sequencing was performed to identify the genetic variants of a Chinese boy who was characterized by skin hyperextensibility, abnormal scarring, hypermobile joints and scoliosis. Sanger sequencing was used to validate the variants in his parents. Reverse transcription polymerase chain reaction (RT-PCR) was performed to analyze the functional effects of the variant. RESULTS: A de novo heterozygous synonymous variant (NM_000393.5:c.1977 G>A) of COL5A2 gene was identified in the patient. The results of RT-PCR revealed that the synonymous variant led to skipping of exon 29 in the RNA transcript. CONCLUSIONS: Our study supplies further supporting evidence that the synonymous COL5A2 mutation c.1977 G>A can cause skipping of exon 29 in the RNA transcript, thus resulting in the production of mutant α2(V)-chains and clinical phenotype of cEDS. This result highlights the need to include splicing-altering synonymous mutations into the screening for cEDS.
Assuntos
Colágeno Tipo V/genética , Síndrome de Ehlers-Danlos/genética , Criança , Colágeno Tipo V/metabolismo , Síndrome de Ehlers-Danlos/patologia , Heterozigoto , Humanos , Masculino , Mutação , Splicing de RNA , Sequenciamento do ExomaRESUMO
BACKGROUND: Gastric cancer (GC) metastasis determines the prognosis of patients, and exploring the molecular mechanism of GC metastasis is expected to provide a theoretical basis for clinical treatment. Recent studies have shown that extracellular matrix protein is closely related to GC metastasis. The present study aimed to explore the expression profile and role of COL5A2, as an extracellular matrix protein, in GC. METHODS: The expression, overall survival, and progression-free survival data of COL5 family members were extracted from The Cancer Genome Atlas (TCGA) database, respectively. Weighted gene co-expression network analysis of the GSE62229 database was performed out to identify modules and associated genes. RESULTS: COL5A2 was selected as our research target in the TCGA database, and was also verified in the GSE62229 and GSE15459 datasets. COL5A2 was up-regulated in GC tissues by paraffin immunohistochemistry and RT-qPCR. The prognosis of patients with low COL5A2 expression was better than that of patients with high COL5A2 expression. Scratch and migration experiments showed that knockdown of COL5A2 decreased the migration ability of gastric cancer cells compared with the control group. In vivo, mice with tail vein injection COL5A2 knockdown had fewer and smaller metastatic nodules in liver. GSEA results showed that the TCGA and GSE62229 samples were significantly enriched in several well-known cancer-related pathways, such as the TGF-ß, MAPK, and JAK2 signaling pathways. CONCLUSION: COL5A2 was most closely related to advanced GC among COL5 family members. High COL5A2 expression is associated with a poor prognosis, and may be a novel therapeutic target for GC.
Assuntos
Biomarcadores Tumorais/genética , Colágeno Tipo V/genética , Neoplasias Gástricas/genética , Animais , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Colágeno Tipo V/metabolismo , Feminino , Humanos , Janus Quinase 2/metabolismo , Sistema de Sinalização das MAP Quinases , Masculino , Camundongos , Pessoa de Meia-Idade , Metástase Neoplásica , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Análise de Sobrevida , Fator de Crescimento Transformador beta/metabolismo , Regulação para CimaRESUMO
OBJECTIVE: The pulmonary vascular remodeling in systemic sclerosis (SSc) is poorly understood and animal models are lacking. Type V collagen (COLV) is elevated in SSc and is implicated in the pathogenesis, and immunization with human COLV induces SSc-like skin and lung changes in rabbits and mice. Here we tested the hypothesis that COLV immunization will induce pathological and functional changes that phenocopy SSc-associated pulmonary vascular disease. METHODS: Pulmonary vascular changes in rabbits immunized with human COLV were extensively characterized by a combination of histology, electron microscopy and immunohistochemistry. Physiologic changes induced by COLV in explanted pulmonary artery rings were evaluated. The pattern of histopathologic alterations and gene expression induced in immunized rabbits were compared to those in SSc patients. RESULTS: COLV immunization was accompanied by striking pulmonary vascular abnormalities, characterized by reduced capillary density, perivascular inflammation, endothelial cell injury and collagen accumulation, that closely phenocopy changes seen in SSc patients. Moreover, pulmonary arteries from immunized rabbits showed impaired ex vivo vascular relaxation. Expression of COL5A2 was significantly increased in the lungs from immunized rabbits (p = 0.02), as well as in patients with SSc (P = 0.02). CONCLUSION: COLV immunity in rabbits is associated with marked vascular remodeling in the lung that phenocopies early-stage human SSc-associated pulmonary vascular disease. COLV immunization therefore represents a novel approach to model SSc pulmonary vascular pathology. Moreover, our findings suggest that COLV might represent a novel pathogenic autoantigen in SSc and future studies with the present model should be developed for possible association with PAH.
Assuntos
Colágeno Tipo V/imunologia , Pulmão/irrigação sanguínea , Artéria Pulmonar/patologia , Escleroderma Sistêmico/patologia , Remodelação Vascular , Adulto , Animais , Estudos de Casos e Controles , Colágeno Tipo V/metabolismo , Modelos Animais de Doenças , Feminino , Hemodinâmica , Humanos , Pessoa de Meia-Idade , Artéria Pulmonar/imunologia , Artéria Pulmonar/metabolismo , Artéria Pulmonar/fisiopatologia , Coelhos , Escleroderma Sistêmico/imunologia , Escleroderma Sistêmico/metabolismo , Escleroderma Sistêmico/fisiopatologiaRESUMO
Ovarian cancer (OC) remains the leading cause of cancer-related death among gynecological cancers. The present study examined the role of collagen type V alpha 1 (COL5A1) and the characteristics of COL5A1 as an oncogenic protein in OC. The association of COL5A1 with paclitaxel (PTX)-resistance and stemness in OC was also studied and the multidatabase and big data analyses of the prognostic value, coexpression network, genetic alterations, and tumor-infiltrating immune cells of COL5A1 were elucidated. We found that COL5A1 expression was high in OC cells and tissues. Knockdown of COL5A1 inhibited the proliferation and migration of OC cells. Further study also showed that COL5A1 was overexpressed in PTX-resistant OC cells compared to respective PTX-sensitive cells. Additionally, COL5A1 was more enriched in OC stem cell-like cells. Silencing COL5A1 expression decreased the OC cell resistance to PTX and inhibited the ability of OC-spheroid formation. Survival analysis predicted that the elevated COL5A1 expression was associated with a worse survival outcome and correlated to the tumor stage of OC patients. The estimating relative subsets of RNA transcripts (CIBERSORT) algorithm analysis also unveiled the correlation of several tumor-infiltrating immune cells with the expression of COL5A1. Taken together, our data demonstrate that COL5A1 is a biomarker to predict OC progression and PTX-resistance and represents a promising target for OC treatment.
Assuntos
Antineoplásicos/farmacologia , Colágeno Tipo V/metabolismo , Linfócitos do Interstício Tumoral/imunologia , Neoplasias Ovarianas/metabolismo , Paclitaxel/farmacologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Colágeno Tipo V/genética , Bases de Dados Genéticas , Progressão da Doença , Resistencia a Medicamentos Antineoplásicos , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Linfócitos do Interstício Tumoral/metabolismo , Invasividade Neoplásica , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/imunologia , Neoplasias Ovarianas/patologia , Microambiente Tumoral , Regulação para CimaRESUMO
Collagen is the most abundant protein in humans. It has a characteristic triple-helix structure and is heavily posttranslationally modified. The complex biosynthesis of collagen involves processing by many enzymes and chaperones in the rough endoplasmic reticulum. Lysyl hydroxylase 1 (LH1) is required to hydroxylate lysine for cross-linking and carbohydrate attachment within collagen triple helical sequences. Additionally, a recent study of prolyl 3-hydroxylase 3 (P3H3) demonstrated that this enzyme may be critical for LH1 activity; however, the details surrounding its involvement remain unclear. If P3H3 is an LH1 chaperone that is critical for LH1 activity, P3H3 and LH1 null mice should display a similar deficiency in lysyl hydroxylation. To test this hypothesis, we compared the amount and location of hydroxylysine in the triple helical domains of type V and I collagen from P3H3 null, LH1 null, and wild-type mice. The amount of hydroxylysine in type V collagen was reduced in P3H3 null mice, but surprisingly type V collagen from LH1 null mice contained as much hydroxylysine as type V collagen from wild-type mice. In type I collagen, our results indicate that LH1 plays a global enzymatic role in lysyl hydroxylation. P3H3 is also involved in lysyl hydroxylation, particularly at cross-link formation sites, but is not required for all lysyl hydroxylation sites. In summary, our study suggests that LH1 and P3H3 likely have two distinct mechanisms to recognize different collagen types and to distinguish cross-link formation sites from other sites in type I collagen.
Assuntos
Colágeno Tipo I/metabolismo , Colágeno Tipo V/metabolismo , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase/metabolismo , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Animais , Colágeno/genética , Colágeno/metabolismo , Colágeno Tipo I/genética , Colágeno Tipo V/genética , Retículo Endoplasmático Rugoso/metabolismo , Hidroxilação , Hidroxilisina/metabolismo , Lisina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pró-Colágeno-Prolina Dioxigenase/genética , Conformação Proteica , Processamento de Proteína Pós-Traducional/genéticaRESUMO
Exosomes isolated from plasma of lung transplant recipients with allograft injury contain donor-derived lung self-antigens (collagen V and Kα1 tubulin) and human leukocyte antigen (HLA) molecules. We present a case of a 76-year-old, female lung transplant recipient treated for acute cellular rejection with methylprednisolone and anti-thymocyte globulin, who subsequently contracted SARS-CoV-2 and developed a sharp increase in the mean fluorescent intensity of anti-HLA antibodies. Analysis of circulating exosomes during rejection, but before SARS-CoV-2 infection, revealed the presence of lung self-antigens and HLA class II molecules. After the patient contracted SARS-CoV-2, exosomes with the SARS-CoV-2 spike protein were also found. After resolution of infectious symptoms, exosomes with SARS-CoV-2 spike protein were no longer detected; however, exosomes with lung self-antigens and HLA class II molecules persisted, which coincided with a progressive decline in spirometric flows, suggesting chronic lung allograft dysfunction. We propose that the analysis of circulating exosomes may be used to detect allograft injury mediated by both rejection and infection. Furthermore, the detection of exosomes containing viral proteins may be helpful in identifying allograft injury driven by viral pathogens.
Assuntos
COVID-19/metabolismo , Exossomos/metabolismo , Rejeição de Enxerto/tratamento farmacológico , Antígenos de Histocompatibilidade Classe II/metabolismo , Imunossupressores/efeitos adversos , Transplante de Pulmão , Glicoproteína da Espícula de Coronavírus/metabolismo , Idoso , Soro Antilinfocitário/uso terapêutico , Autoantígenos/imunologia , Autoantígenos/metabolismo , Bronquiolite Obliterante , COVID-19/imunologia , Colágeno Tipo V/imunologia , Colágeno Tipo V/metabolismo , Progressão da Doença , Feminino , Glucocorticoides/efeitos adversos , Glucocorticoides/uso terapêutico , Antígenos HLA/imunologia , Antígenos HLA/metabolismo , Antígenos de Histocompatibilidade Classe II/imunologia , Humanos , Imunossupressores/uso terapêutico , Metilprednisolona/efeitos adversos , Metilprednisolona/uso terapêutico , SARS-CoV-2/imunologia , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/imunologia , Tubulina (Proteína)/imunologia , Tubulina (Proteína)/metabolismoRESUMO
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.