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1.
Sci Adv ; 6(31)2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32937590

RESUMO

The outbreak of the highly contagious and deadly severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), also known as coronavirus disease 2019 (COVID-19), has posed a serious threat to public health across the globe, calling for the development of effective diagnostic markers and therapeutics. Here, we report a highly reliable severity diagnostic biomarker, acetylated 676th lysine transforming growth factor-beta-induced protein (TGFBIp K676Ac). TGFBIp K676Ac was consistently elevated in the blood of patients with SARS-CoV-2 pneumonia (n = 113), especially in patients in the intensive care unit (ICU) compared to non-ICU patients. Patients' blood samples showed increased cytokines and lymphopenia, which are exemplary indicators of SARS-CoV-2 pneumonia. Treatment with TGFBIp neutralizing antibodies suppressed the cytokine storm. The increased level of TGFBIp K676Ac in ICU patients suggests the promise of this protein as a reliable severity diagnostic biomarker for severe SARS-CoV-2 disease.


Assuntos
Betacoronavirus/patogenicidade , Infecções por Coronavirus/diagnóstico , Síndrome da Liberação de Citocina/diagnóstico , Proteínas da Matriz Extracelular/imunologia , Leucócitos Mononucleares/imunologia , Pneumonia Viral/diagnóstico , Processamento de Proteína Pós-Traducional , Insuficiência Respiratória/diagnóstico , Fator de Crescimento Transformador beta/imunologia , Acetilação , Anticorpos Neutralizantes/farmacologia , Betacoronavirus/imunologia , Biomarcadores/sangue , Estudos de Casos e Controles , Infecções por Coronavirus/sangue , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/patologia , Síndrome da Liberação de Citocina/sangue , Síndrome da Liberação de Citocina/imunologia , Síndrome da Liberação de Citocina/patologia , Proteínas da Matriz Extracelular/antagonistas & inibidores , Proteínas da Matriz Extracelular/genética , Expressão Gênica , Humanos , Unidades de Terapia Intensiva , Contagem de Leucócitos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/patologia , Leucócitos Mononucleares/virologia , Pulmão/irrigação sanguínea , Pulmão/efeitos dos fármacos , Pulmão/patologia , Pulmão/virologia , Lisina/metabolismo , NF-kappa B/genética , NF-kappa B/imunologia , Pandemias , Pneumonia Viral/sangue , Pneumonia Viral/imunologia , Pneumonia Viral/patologia , Cultura Primária de Células , Prognóstico , Insuficiência Respiratória/sangue , Insuficiência Respiratória/imunologia , Insuficiência Respiratória/patologia , Índice de Gravidade de Doença , Fator de Crescimento Transformador beta/antagonistas & inibidores , Fator de Crescimento Transformador beta/genética
2.
Gene ; 761: 145024, 2020 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-32755659

RESUMO

Understanding how various pathologies of breast cancer respond to their environment may be imperative in the creation of novel therapeutic targets. Central to the organisation and behaviour of cells within the tumour microenvironment is the extracellular matrix (ECM), a meshwork of fibrous proteins and glycoproteins that directly influences cell behaviour and the bioavailability of signalling molecules. Our appreciation on how the composition of the ECM can influence cancer behaviour has evolved significantly and although we are highly cognisant of the dramatic impact the ECM can have on cancer cell behaviour, we continue to neglect this during diagnosis and treatment. In the following study, we aimed to identify how three breast cancer cell lines respond functionally and genetically to common components of the ECM. Using real time and end point assays we have identified similar patterns of behaviour among the three breast cancer cell lines in response to commonly found ECM components of the breast. Using a selected gene panel, we have been able to identify cell line specific changes in gene differentiation when breast cancer cells are in contact with these elements. Although the response of our cells to these elements differ at the genetic level, their functional responses are consistent. This work adds to the growing arguments that highlight a need for histologically assessing ECM composition of breast tumours. In particular monitoring of fibrous protein deposition at the site of malignancy could provide critical information during clinical assessment influencing disease prognosis and treatment decisions for breast cancer patients.


Assuntos
Neoplasias da Mama/genética , Colágeno/genética , Fibronectinas/genética , Mama/patologia , Linhagem Celular Tumoral , Colágeno/metabolismo , Colágeno Tipo I/genética , Matriz Extracelular/metabolismo , Proteínas da Matriz Extracelular/genética , Feminino , Fibronectinas/metabolismo , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica/genética , Genótipo , Glicoproteínas/genética , Humanos , Fenótipo , Prognóstico , Transdução de Sinais , Microambiente Tumoral
3.
Rev Assoc Med Bras (1992) ; 66(5): 680-686, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32638975

RESUMO

OBJECTIVE Pelvic organ prolapse (POP) is a very frequent situation in our population that may lead to a significant decrease in patients' quality of life. Currently, we are looking for predictive factors for the development of POPs; thus, this study seeks to evaluate whether the Fibulin 5 polymorphism (FBLN5) is associated with the occurrence of POP. METHODS This is a cohort study with postmenopausal women who were divided into groups by POP stage: POP stages 0 and I (control group) and POP stages III and IV (case group). Subsequently, analyses of genetic polymorphisms of FBLN5 were performed using the Restriction Fragment Length Polymorphism (RFLP) technique. RESULTS A total of 292 women were included in the study. Pregnancy, parity and vaginal delivery in the patients, as well as in data described in the literature, were related to the occurrence of POP in the univariate analysis. However, after binary logistic regression, home birth and age remained independent risk factors for POP. We found no association between the FBLN5 polymorphism and the occurrence of POP (p = 0.371). CONCLUSION There was no association between the FBLN5 polymorphism and the occurrence of POP in Brazilian women.


Assuntos
Proteínas da Matriz Extracelular/genética , Prolapso de Órgão Pélvico , Qualidade de Vida , Brasil , Proteínas de Ligação ao Cálcio/genética , Estudos de Coortes , Feminino , Humanos , Polimorfismo Genético , Gravidez
4.
Science ; 369(6499): 71-77, 2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32527924

RESUMO

Bacterial biofilms represent a basic form of multicellular organization that confers survival advantages to constituent cells. The sequential stages of cell ordering during biofilm development have been studied in the pathogen and model biofilm-former Vibrio cholerae It is unknown how spatial trajectories of individual cells and the collective motions of many cells drive biofilm expansion. We developed dual-view light-sheet microscopy to investigate the dynamics of biofilm development from a founder cell to a mature three-dimensional community. Tracking of individual cells revealed two distinct fates: one set of biofilm cells expanded ballistically outward, while the other became trapped at the substrate. A collective fountain-like flow transported cells to the biofilm front, bypassing members trapped at the substrate and facilitating lateral biofilm expansion. This collective flow pattern was quantitatively captured by a continuum model of biofilm growth against substrate friction. Coordinated cell movement required the matrix protein RbmA, without which cells expanded erratically. Thus, tracking cell lineages and trajectories in space and time revealed how multicellular structures form from a single founder cell.


Assuntos
Biofilmes , Vibrio cholerae/citologia , Vibrio cholerae/fisiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Microscopia , Movimento (Física) , Mutação , Análise de Célula Única/métodos , Vibrio cholerae/genética
5.
PLoS One ; 15(6): e0235122, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32584873

RESUMO

The MGP single nucleotide polymorphism (SNP) rs1800801 has previously been associated with recurrent ischemic stroke in a Spanish cohort. Here, we tested for association of this SNP with ischemic stroke recurrence in a North American Caucasian cohort. Acute ischemic stroke patients admitted between 10/2009 and 12/2016 at three hospitals within a large healthcare system in the northeastern United States that were enrolled in a healthcare system-wide exome sequencing program were retrospectively reviewed. Patients with recurrent stroke within 1 year after index event were compared to those without recurrence. Of 9,348 suspected acute ischemic strokes admitted between 10/2009 and 12/2016, 1,727 (18.5%) enrolled in the exome-sequencing program. Among those, 1,068 patients had exome sequencing completed and were eligible for inclusion. Recurrent stroke within the first year of stroke was observed in 79 patients (7.4%). In multivariable analysis, stroke prior to the index stroke (OR 9.694, 95% CI 5.793-16.224, p ≤ 0.001), pro-coagulant status (OR = 3.563, 95% CI 1.504-8.443, p = 0.004) and the AA genotype of SNP rs1800801 (OR = 2.408, 95% CI 1.079-4.389, p = 0.004) were independently associated with recurrent stroke within the first year. The AA genotype of the MGP SNP rs1800801 is associated with recurrence within the first year after ischemic stroke in North American Caucasians. Study of stroke subtypes and additional populations will be required to determine if incorporation of allelic status at this SNP into current risk scores improves prediction of recurrent ischemic stroke.


Assuntos
Isquemia Encefálica/genética , Proteínas de Ligação ao Cálcio/genética , Proteínas da Matriz Extracelular/genética , Predisposição Genética para Doença , Genótipo , Polimorfismo de Nucleotídeo Único , Acidente Vascular Cerebral/genética , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Recidiva , Fatores de Risco , Sequenciamento Completo do Exoma
6.
Prostate ; 80(10): 753-763, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32421868

RESUMO

BACKGROUND: Although thrombospondins 4 (THBS4) participates in controlling the biology of prostate cancer (PCa), the mechanism underlying this regulation remains unknown. Hence, this study aims to identify the regulatory effects of THBS4 on the PCa stem cell-like properties and the potential mechanism associated with the phosphatidylinositol 3'-kinase (PI3K)/protein kinase B (Akt) pathway. METHODS: PCa stem cells were sorted and identified using flow cytometry and THBS4 expression in the identified PCa stem cells was measured using Western blot assay. THBS4 was overexpressed or silenced in PCa stem cells, following which, self-renewal, proliferation, cell cycle distribution, and apoptosis of PCa stem cells were assessed as well as tumorigenicity in vivo was evaluated. PI3K/Akt pathway inhibitor was applied to identify its involvement in the regulatory roles of THBS4 in PCa stem cells. RESULTS: THBS4 was expressed at a higher level in PCa stem cells than in PCa cells. The overexpression of THBS4 promoted the self-renewal and proliferation, curbed the apoptosis of PCa stem cells, and enhanced the in vivo tumorigenicity, which was achieved by activating the PI3K/Akt pathway. On the contrary, short-hairpin RNA-mediated silencing of THBS4 exhibited suppressive effects on those cancer stem cell (CSC)-like properties and promotive effects on their apoptosis. CONCLUSION: THBS4 silencing can impede the CSC-like properties in PCa via blockade of the PI3K/Akt pathway, which provides patients with PCa a new therapeutic target.


Assuntos
Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Trombospondinas/metabolismo , Antígeno AC133/biossíntese , Animais , Linhagem Celular Tumoral , Proteínas da Matriz Extracelular/biossíntese , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Perfilação da Expressão Gênica , Inativação Gênica , Xenoenxertos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos Nus , Camundongos SCID , Células PC-3 , Neoplasias da Próstata/genética , Transdução de Sinais , Trombospondinas/biossíntese , Trombospondinas/deficiência , Trombospondinas/genética
7.
PLoS One ; 15(5): e0232779, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32365083

RESUMO

Apoptosis of neurovascular cells, including astroglial cells, contributes to the pathogenesis of diseases in which neurovascular disruption plays a central role. Bim is a pro-apoptotic protein that modulates not only apoptosis but also various cellular functions such as migration and extracellular matrix protein expression. Astroglial cells act as an intermediary between neural and vascular cells facilitating retinal vascular development and remodeling while maintaining normal vascular function and neuronal integrity. We previously showed that Bim deficient (Bim -/-) mice were protected from hyperoxia mediated vessel obliteration and ischemia-mediated retinal neovascularization. However, the underlying mechanisms and more specifically the role Bim expression in astroglial cells play remains elusive. Here, using retinal astroglial cells prepared from wild-type and Bim -/- mice, we determined the impact of Bim expression in retinal astroglial cell function. We showed that astroglial cells lacking Bim expression demonstrate increased VEGF expression and altered matricellular protein production including increased expression of thrombospondin-2 (TSP2), osteopontin and SPARC. Bim deficient astroglial cells also exhibited altered proliferation, migration, adhesion to various extracellular matrix proteins and increased expression of inflammatory mediators. Thus, our data emphasizes the importance of Bim expression in retinal astroglia cell autonomous regulatory mechanisms, which could influence neurovascular function.


Assuntos
Astrócitos/metabolismo , Proteína 11 Semelhante a Bcl-2/genética , Inflamação/genética , Retina/metabolismo , Animais , Apoptose/genética , Astrócitos/patologia , Movimento Celular/genética , Proliferação de Células/genética , Proteínas da Matriz Extracelular/genética , Regulação da Expressão Gênica/genética , Humanos , Inflamação/metabolismo , Inflamação/patologia , Camundongos , Neovascularização Fisiológica/genética , Neurônios/metabolismo , Osteonectina/genética , Osteopontina/genética , Retina/patologia , Trombospondinas/genética , Fator A de Crescimento do Endotélio Vascular/genética
8.
PLoS One ; 15(5): e0233422, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32437418

RESUMO

SPARCL1 is a matricellular protein with anti-adhesive, anti-proliferative and anti-tumorigenic functions and is frequently downregulated in tumors such as colorectal carcinoma or non-small cell lung cancer. Studies have identified SPARCL1 as an angiocrine tumor suppressor secreted by tumor vessel endothelial cells, thereby exerting inhibitory activity on angiogenesis and tumor growth, in colorectal carcinoma. It is unknown whether SPARCL1 may exert these homeostatic functions in all organs and in other species. Therefore, SPARCL1 expression was comparatively analysed between humans and mice in a systematic manner. Murine Sparcl1 (mSparcl1) is most strongly expressed in the lung; expressed at an intermediate level in most organs, including the large intestine; and absent in the liver. In human tissues, SPARCL1 (hSPARCL1) was detected in all organs, with the strongest expression in the stomach, large intestine and lung, mostly consistent with the murine expression pattern. A striking difference between human and murine tissues was the absence of mSparcl1 expression in murine livers, while human livers showed moderate expression. Furthermore, mSparcl1 was predominantly associated with mural cells, whereas hSPARCL1 was detected in both mural and endothelial cells. Human SPARCL1 expression was downregulated in different carcinomas, including lung and colon cancers. In conclusion, this study revealed species-, organ- and cell-type-dependent expression of SPARCL1, suggesting that its function may not be similar between humans and mice.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Mucosa Intestinal/metabolismo , Fígado/metabolismo , Pulmão/metabolismo , Animais , Proteínas de Ligação ao Cálcio/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Proteínas da Matriz Extracelular/genética , Mucosa Gástrica , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Knockout , Especificidade de Órgãos , Especificidade da Espécie
9.
Hum Genet ; 139(10): 1315-1323, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32382995

RESUMO

We present detailed comparative analyses to assess population-level differences in patterns of genetic deafness between European/American and Japanese cohorts with non-syndromic hearing loss. One thousand eighty-three audiometric test results (921 European/American and 162 Japanese) from members of 168 families (48 European/American and 120 Japanese) with non-syndromic hearing loss secondary to pathogenic variants in one of three genes (KCNQ4, TECTA, WFS1) were studied. Audioprofile characteristics, specific mutation types, and protein domains were considered in the comparative analyses. Our findings support differences in audioprofiles driven by both mutation type (non-truncating vs. truncating) and ethnic background. The former finding confirms data that ascribe a phenotypic consequence to different mutation types in KCNQ4; the latter finding suggests that there are ethnic-specific effects (genetic and/or environmental) that impact gene-specific audioprofiles for TECTA and WFS1. Identifying the drivers of ethnic differences will refine our understanding of phenotype-genotype relationships and the biology of hearing and deafness.


Assuntos
Proteínas da Matriz Extracelular/genética , Genótipo , Perda Auditiva Neurossensorial/genética , Canais de Potássio KCNQ/genética , Proteínas de Membrana/genética , Mutação , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Grupo com Ancestrais do Continente Asiático , Audiometria , Estudos de Casos e Controles , Criança , Pré-Escolar , Grupo com Ancestrais do Continente Europeu , Feminino , Proteínas Ligadas por GPI/genética , Expressão Gênica , Estudos de Associação Genética , Perda Auditiva Neurossensorial/diagnóstico , Perda Auditiva Neurossensorial/etnologia , Perda Auditiva Neurossensorial/fisiopatologia , Humanos , Lactente , Recém-Nascido , Japão , Masculino , Pessoa de Meia-Idade , Linhagem , Fenótipo , Estados Unidos
10.
Nat Cell Biol ; 22(7): 882-895, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32451439

RESUMO

It is well accepted that cancers co-opt the microenvironment for their growth. However, the molecular mechanisms that underlie cancer-microenvironment interactions are still poorly defined. Here, we show that Rho-associated kinase (ROCK) in the mammary tumour epithelium selectively actuates protein-kinase-R-like endoplasmic reticulum kinase (PERK), causing the recruitment and persistent education of tumour-promoting cancer-associated fibroblasts (CAFs), which are part of the cancer microenvironment. An analysis of tumours from patients and mice reveals that cysteine-rich with EGF-like domains 2 (CRELD2) is the paracrine factor that underlies PERK-mediated CAF education downstream of ROCK. We find that CRELD2 is regulated by PERK-regulated ATF4, and depleting CRELD2 suppressed tumour progression, demonstrating that the paracrine ROCK-PERK-ATF4-CRELD2 axis promotes the progression of breast cancer, with implications for cancer therapy.


Assuntos
Neoplasias da Mama/patologia , Fibroblastos Associados a Câncer/patologia , Moléculas de Adesão Celular/metabolismo , Reprogramação Celular , Proteínas da Matriz Extracelular/metabolismo , eIF-2 Quinase/metabolismo , Quinases Associadas a rho/metabolismo , Fator 4 Ativador da Transcrição/genética , Fator 4 Ativador da Transcrição/metabolismo , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Fibroblastos Associados a Câncer/metabolismo , Moléculas de Adesão Celular/genética , Células Cultivadas , Modelos Animais de Doenças , Retículo Endoplasmático/metabolismo , Proteínas da Matriz Extracelular/genética , Feminino , Humanos , Camundongos , Comunicação Parácrina , eIF-2 Quinase/genética , Quinases Associadas a rho/genética
11.
Am J Physiol Renal Physiol ; 318(6): F1478-F1488, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32390515

RESUMO

Activation of immunological pathways and disturbances of extracellular matrix (ECM) dynamics are important contributors to the pathogenesis of chronic kidney diseases. Glomerular mesangial cells (MCs) are critical for homeostasis of glomerular ECM dynamics. Interleukin-6 (IL-6) can act as a pro/anti-inflammatory agent relative to cell types and conditions. This study investigated whether IL-6 influences ECM protein production by MCs and the regulatory pathways involved. Experiments were carried out in cultured human MCs (HMCs) and in mice. We found that overexpression of IL-6 and its receptor decreased the abundance of fibronectin and collagen type IV in MCs. ELISA and immunoblot analysis demonstrated that thapsigargin [an activator of store-operated Ca2+ entry (SOCE)], but not the endoplasmic reticulum stress inducer tunicamycin, significantly increased IL-6 content. This thapsigargin effect was abolished by GSK-7975A, a selective inhibitor of SOCE, and by silencing Orai1 (the channel protein mediating SOCE). Furthermore, inhibition of NF-κB pharmacologically and genetically significantly reduced SOCE-induced IL-6 production. Thapsigargin also stimulated nuclear translocation of the p65 subunit of NF-κB. Moreover, MCs overexpressing IL-6 and its receptor in HMCs increased the content of the glucagon-like peptide-1 receptor (GLP-1R), and IL-6 inhibition of fibronectin was attenuated by the GLP-1R antagonist exendin 9-39. In agreement with the HMC data, specific knockdown of Orai1 in MCs using the targeted nanoparticle delivery system in mice significantly reduced glomerular GLP-1R levels. Taken together, our results suggest a novel SOCE/NF-κB/IL-6/GLP-1R signaling pathway that inhibits ECM protein production by MCs.


Assuntos
Proteínas da Matriz Extracelular/metabolismo , Interleucina-6/metabolismo , Células Mesangiais/metabolismo , Receptores de Interleucina-6/metabolismo , Animais , Células Cultivadas , Regulação para Baixo , Proteínas da Matriz Extracelular/genética , Receptor do Peptídeo Semelhante ao Glucagon 1/genética , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Humanos , Interleucina-6/genética , Masculino , Camundongos Endogâmicos C57BL , NF-kappa B/genética , NF-kappa B/metabolismo , Proteína ORAI1/genética , Proteína ORAI1/metabolismo , Receptores de Interleucina-6/genética , Transdução de Sinais
12.
Invest Ophthalmol Vis Sci ; 61(4): 24, 2020 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-32305042

RESUMO

Purpose: To identify processes that contribute to corticosteroid-induced ocular hypertension and candidate target genes for treatment. Methods: A systematic search identified five human microarray datasets investigating the effect of dexamethasone versus a control medium on trabecular meshwork (TM) tissue. After thorough quality control, samples of low quality were removed, and the datasets were integrated. Additionally, a bovine RNA-sequencing dataset allowed to investigate differences in gene expression profiling between cows with and without corticosteroid-induced ocular hypertension (responders vs. nonresponders). The obtained datasets were used as input for parallel pathway analyses. Significantly changed pathways were clustered into functional categories and the results were further investigated. A network visualizing the differences between the responders and nonresponders was created. Results: Seven functional pathway clusters were found to be significantly changed in TM cells exposed to dexamethasone versus a control medium and in TM cells of responders versus nonresponders: collagen, extracellular matrix, adhesion, WNT-signaling, inflammation, adipogenesis, and glucose metabolism. In addition, cell cycle and senescence were only significantly changed in responders versus nonresponders. The network of the differential gene expression between responders and nonresponders shows many connections between the identified processes via shared genes. Conclusions: Nine functional pathway clusters synthesize the molecular response to dexamethasone exposure in TM cells and are likely to be involved in the pathogenesis of corticosteroid-induced ocular hypertension.


Assuntos
Proteínas de Ciclo Celular/genética , Dexametasona/farmacologia , Proteínas da Matriz Extracelular/genética , Regulação da Expressão Gênica/fisiologia , Glucocorticoides/farmacologia , Hipertensão Ocular/induzido quimicamente , Malha Trabecular/efeitos dos fármacos , Animais , Bovinos , Células Cultivadas , Conjuntos de Dados como Assunto , Perfilação da Expressão Gênica , Humanos , Hipertensão Ocular/genética , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Malha Trabecular/metabolismo
13.
J Cancer Res Clin Oncol ; 146(6): 1509-1521, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32266537

RESUMO

PURPOSE: It is important for hepatocellular carcinoma (HCC) treatment that the targets related to its progression are identified. Clustered regularly interspaced short palindromic repeat (CRISPR)-associated nuclease 9 (Cas9)-based genetic screening is a powerful tool for identifying genes with loss-of-function mutations that are critical for tumour growth and metastasis. METHODS: We transduced the human SMMC7721 HCC cell line expressing Cas9 with a human genome-scale CRISPR-Cas9 knockout (GeCKO) lentiviral library A (hGeCKOa) of 65,383 single-guide RNAs (sgRNAs) targeting 19,050 human genes; we then subcutaneously transplanted the transduced cells into nude mice. RESULTS: The transduced cells were found to proliferate and metastasize faster than the untransduced cells. Through next-generation sequencing, the genes potentially related to HCC proliferation and metastasis were identified. The sgRNAs targeting the ADAMTSL3 and PTEN genes appeared twice on the list of genes related to HCC proliferation and metastasis, respectively. Analysis based on the data mining of Oncomine revealed that the ADAMTSL3 and PTEN genes were expressed at lower levels in HCC cells than they were in normal liver cells, indicating their tumour-suppressive roles. Downregulation of ADAMTSL3 and PTEN displayed poor overall survival (OS) and predicted poor relapse-free survival (RFS), further supporting their tumour-suppressive roles. Moreover, knocking out either the ADAMTSL3 or PTEN genes promoted either the proliferation or metastasis of HCC cells, respectively. CONCLUSIONS: Using both in vitro and in vivo approaches, we described the profound role of the ADAMTSL3 and PTEN genes. This study indicates novel candidate targets for use in HCC treatment and therapy.


Assuntos
Proteínas ADAMTS/genética , Carcinoma Hepatocelular/patologia , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Proteínas da Matriz Extracelular/genética , Neoplasias Hepáticas/patologia , PTEN Fosfo-Hidrolase/genética , Animais , Carcinoma Hepatocelular/genética , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Neoplasias Hepáticas/genética , Masculino , Camundongos , Camundongos Endogâmicos , Camundongos Nus , Metástase Neoplásica
14.
J Neurosci ; 40(20): 4059-4072, 2020 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-32265257

RESUMO

Photoreceptor neurons are surrounded by an extracellular matrix, called the interphotoreceptor matrix (IPM). Activities crucial to vision occur within the IPM, including trafficking of nutrients and metabolites, retinal attachment, and interactions needed for normal outer segment phagocytosis. The IPM includes the following two unique proteoglycans: IPM proteoglycan 1 (IMPG1) and IMPG2. Patients with mutations in IMPG1/IMPG2 develop visual deficits with subretinal material accumulation, highlighting the critical role of the IPM in vision. To determine the role of these proteoglycans in retinal physiology and the pathologic mechanisms that lead to vision loss, we generated mouse models lacking IMPG1/IMPG2. In normal retina, IMPG1 and IMPG2 occupy distinct IPM compartments, represent the main source of chondroitin sulfate and are fundamental for the constitution of the cone-specific glycocalyx stained by the PNA (peanut agglutinin) lectin marker. No evident morphologic or functional deficits were found in mice lacking IMPG1. In the absence of IMPG2, IMPG1 abnormally accumulated at the subretinal space need, likely leading to the formation of subretinal lesions and reduced visual function. Interestingly, mice lacking both IMPG1 and IMPG2, regardless of sex, showed normal retinal structure and function, demonstrating that the aberrant IMPG1 distribution is the main cause of the visual alterations observed in the absence of IMPG2. In conclusion, our results show the dependence of secreted proteoglycans such as IMPG1 on the extracellular environment to properly integrate into the matrix, demonstrate the role of IMPG2 in shaping the IPM, and shed light on the potential mechanisms leading to the development of subretinal lesions and vision loss.SIGNIFICANCE STATEMENT The photoreceptors are specialized neurons that drive phototransduction in the mammalian retina. These cells are organized and surrounded by an extracellular matrix, the interphotoreceptor matrix (IPM). Mutations in IPM proteoglycans are associated with blindness in humans. Our studies show that two specific proteoglycans of the IPM, IPM proteoglycan 1 (IMPG1) and IMPG2, form a dynamic structure with distinct localization and dependency. When IMPG2 is absent, IMPG1 cannot integrate into the IPM, leading to abnormal proteoglycan accumulation and visual deficits. This work adds a new layer of understanding to IPM physiology and describes the pathologic events following deficits in proteoglycans, providing novel possibilities for visual restoration in patients with IMPG-related pathologies.


Assuntos
Células Fotorreceptoras de Vertebrados/fisiologia , Proteoglicanas/genética , Visão Ocular/fisiologia , Animais , Sulfatos de Condroitina/metabolismo , Eletrorretinografia , Proteínas da Matriz Extracelular/genética , Espaço Extracelular , Proteínas do Olho/genética , Feminino , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Knockout , Mutação , Retina/fisiopatologia , Células Fotorreceptoras Retinianas Cones/metabolismo , Transtornos da Visão/genética
15.
Proc Natl Acad Sci U S A ; 117(17): 9413-9422, 2020 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-32291340

RESUMO

Astrogenesis is repressed in the early embryonic period and occurs in the late embryonic period. A variety of external and internal signals contribute to the sequential differentiation of neural stem cells. Here, we discovered that immune-related CD93 plays a critical negative role in the regulation of astrogenesis in the mouse cerebral cortex. We show that CD93 expression is detected in neural stem cells and neurons but not in astrocytes and declines as differentiation proceeds. Cd93 knockout increases astrogenesis at the expense of neuron production during the late embryonic period. CD93 responds to the extracellular matrix protein Multimerin 2 (MMRN2) to trigger the repression of astrogenesis. Mechanistically, CD93 delivers signals to ß-Catenin through a series of phosphorylation cascades, and then ß-Catenin transduces these signals to the nucleus to activate Zfp503 transcription. The transcriptional repressor ZFP503 inhibits the transcription of glial fibrillary acidic protein (Gfap) by binding to the Gfap promoter with the assistance of Grg5. Furthermore, Cd93 knockout mice exhibit autism-like behaviors. Taken together, our results reveal that CD93 is a negative regulator of the onset of astrogenesis and provide insight into therapy for psychiatric disorders.


Assuntos
Astrócitos/fisiologia , Proteínas da Matriz Extracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Glicoproteínas de Membrana/metabolismo , Subfamília D de Receptores Semelhantes a Lectina de Células NK/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Animais , Transtorno Autístico , Córtex Cerebral/citologia , Córtex Cerebral/embriologia , Eletroporação , Proteínas da Matriz Extracelular/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Inflamação , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos ICR , Subfamília D de Receptores Semelhantes a Lectina de Células NK/genética , Proteínas do Tecido Nervoso/genética , Neurogênese , Neuroglia , Gravidez
16.
Biol Res ; 53(1): 10, 2020 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-32156314

RESUMO

BACKGROUND: The aim of this study was to investigate the effect role and mechanism of miR-30b-3p on ovarian cancer cells biological function. METHODS: The expression of miR-30b-3p was detected in ovarian cancer cell lines and normal ovarian epithelial cell line by qRT-PCR. Mir-30b-3p mimic was transfected into OVCAR3 cells. Cell-counting kit-8 (CCK-8) assay was conducted to explore the effect of mir-30b-3p on the OVCAR3 cells' proliferation. Cell cycle and apoptosis were detected by Flow cytometry. Cell invasion ability was detected by Transwell test. The regulation of putative target of miR-30b-3p was verified by double luciferase reporter assays and Western blot. RESULT: We found that miR-30b-3p was downregulated in OVCAR3 cells. Overexpression of miR-30b-3p suppressed proliferation, promoted apoptosis, slowed cell cycle and inhibited migration and invasion of OVCAR3 cells. Bioinformatics analysis identified 3'-untranslated region (3'UTR) of Collagen triple helix repeat-containing 1 (CTHRC1) as the presumed binding site for miR-30b-3p. Detection of double luciferase reporter and Western-Blot result confirmed that CTHRC1 was the target gene of miR-30b-3p. Furthermore, E-cadherin, ß-cadherin and Vimentin protein expression level were changed after transfection of miR-30b-3p. CONCLUSION: miR-30b-3p function as an anti-cancer gene. Overexpression of miR-30b-3p can inhibit the biological function of ovarian cancer cells. MiR-30b-3p targets CTHRC1 gene plays an important role in epithelial-mesenchymal transformation (EMT), and supports miR-30b-3p as a potential biological indicator for ovarian cancer in the future.


Assuntos
Transição Epitelial-Mesenquimal/genética , Proteínas da Matriz Extracelular/genética , Regulação Neoplásica da Expressão Gênica/genética , MicroRNAs/genética , Neoplasias Ovarianas/genética , Apoptose , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Proteínas da Matriz Extracelular/metabolismo , Feminino , Humanos , Invasividade Neoplásica , Neoplasias Ovarianas/metabolismo , Transdução de Sinais
18.
Artigo em Inglês | MEDLINE | ID: mdl-32178265

RESUMO

The ENAM gene is important in the formation of tooth enamel; an alteration can affect the lengthening of the crystals, and the thickness in enamel. The objective was to determine the presence of the single nucleotide variant (SNV) rs12640848 of the ENAM gene in students exposed to different concentrations of fluoride. METHODS: A cross-sectional study was conducted on students exposed to high concentrations of fluoride in the city of Durango which were divided according to the severity of fluorosis and dental caries. Genotype determination was performed by DNA sequencing. The relationship between the severity of dental fluorosis and the allele distribution was determined by the Fisher's exact and Kruskal-Wallis tests. RESULTS: Seventy-one students were included for the sequencing. In the different allelic variations, for the normal genotype AA/TT, the control group presented 75%, for the AG/TC variation, 70.8% in the TF ≤ 4 group, 65% in TF ≥ 5, and 16.7% in TF = 0; with respect to GG/CC variation, 12.5% in TF ≤ 4, 22% in TF ≥ 5, and 8.3% in TF = 0 group (p = 0.000). CONCLUSION: The ENAM gene showed an association in the population exposed to different concentrations of fluoride.


Assuntos
Cárie Dentária , Proteínas da Matriz Extracelular , Fluoretos , Fluorose Dentária , Alelos , Estudos Transversais , Cárie Dentária/genética , Proteínas da Matriz Extracelular/genética , Fluorose Dentária/genética , Humanos , Estudantes
19.
J Vis Exp ; (156)2020 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-32116296

RESUMO

Extracellular matrix (ECM) proteins are crucial for skeletal muscle development and homeostasis. The stable knockdown of genes coding for ECM proteins in C2C12 myoblasts can be applied to study the role of these proteins in skeletal muscle development. Here, we describe a protocol to deplete the ECM protein ADAMTSL2 as an example, using small-hairpin (sh) RNA in C2C12 cells. Following transfection of shRNA plasmids, stable cells were batch-selected using puromycin. We further describe the maintenance of these cell lines and the phenotypic analysis via mRNA expression, protein expression, and C2C12 differentiation. The advantages of the method are the relatively fast generation of stable C2C12 knockdown cells and the reliable differentiation of C2C12 cells into multinucleated myotubes upon depletion of serum in the cell culture medium. Differentiation of C2C12 cells can be monitored by bright field microscopy and by measuring the expression levels of canonical marker genes, such as MyoD, myogenin, or myosin heavy chain (MyHC) indicating the progression of C2C12 myoblast differentiation into myotubes. In contrast to the transient knockdown of genes with small-interfering (si) RNA, genes that are expressed later during C2C12 differentiation or during myotube maturation can be targeted more efficiently by generating C2C12 cells that stably express shRNA. Limitations of the method are a variability in the knockdown efficiencies, depending on the specific shRNA that may be overcome by using gene knockout strategies based on CRISPR/Cas9, as well as potential off-target effects of the shRNA that should be considered.


Assuntos
Proteínas ADAMTS/genética , Proteínas da Matriz Extracelular/genética , Técnicas de Silenciamento de Genes , Mioblastos/metabolismo , RNA Interferente Pequeno/genética , Proteínas ADAMTS/metabolismo , Animais , Diferenciação Celular , Linhagem Celular , Proteínas da Matriz Extracelular/metabolismo , Camundongos , Fibras Musculares Esqueléticas/citologia , Mioblastos/citologia , RNA Mensageiro/metabolismo
20.
Lab Invest ; 100(7): 928-944, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32203150

RESUMO

The tumor microenvironment is increasingly recognized as key player in cancer progression. Investigating heterotypic interactions between cancer cells and their microenvironment is important for understanding how specific cell types support cancer. Forming the vasculature, endothelial cells (ECs) are a prominent cell type in the microenvironment of both normal and neoplastic breast gland. Here, we sought out to analyze epithelial-endothelial cross talk in the breast using isogenic non-tumorigenic vs. tumorigenic breast epithelial cell lines and primary ECs. The cellular model used here consists of D492, a breast epithelial cell line with stem cell properties, and two isogenic D492-derived EMT cell lines, D492M and D492HER2. D492M was generated by endothelial-induced EMT and is non-tumorigenic while D492HER2 is tumorigenic, expressing the ErbB2/HER2 oncogene. To investigate cellular cross talk, we used both conditioned medium (CM) and 2D/3D co-culture systems. Secretome analysis of D492 cell lines was performed using mass spectrometry and candidate knockdown (KD), and overexpression (OE) was done using siRNA and CRISPRi/CRISPRa technology. D492HER2 directly enhances endothelial network formation and activates a molecular axis in ECs promoting D492HER2 migration and invasion, suggesting an endothelial feedback response. Secretome analysis identified extracellular matrix protein 1 (ECM1) as potential angiogenic inducer in D492HER2. Confirming its involvement, KD of ECM1 reduced the ability of D492HER2-CM to increase endothelial network formation and induce the endothelial feedback, while recombinant ECM1 (rECM1) increased both. Interestingly, NOTCH1 and NOTCH3 expression was upregulated in ECs upon treatment with D492HER2-CM or rECM1 but not by CM from D492HER2 with ECM1 KD. Blocking endothelial NOTCH signaling inhibited the increase in network formation and the ability of ECs to promote D492HER2 migration and invasion. In summary, our data demonstrate that cancer-secreted ECM1 induces a NOTCH-mediated endothelial feedback promoting cancer progression by enhancing migration and invasion. Targeting this interaction may provide a novel possibility to improve cancer treatment.


Assuntos
Neoplasias da Mama/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Invasividade Neoplásica/genética , Receptor ErbB-2/metabolismo , Microambiente Tumoral/genética , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Proteínas da Matriz Extracelular/genética , Feminino , Humanos , Receptor ErbB-2/genética
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