RESUMO
BACKGROUND This study aimed to investigate the effects of microRNA-515-5p (miR-515-5p) on the expression of the WNT1-inducible-signaling pathway protein 1 (WISP-1) gene in rheumatoid arthritis fibroblast-like synovial (RAFLS) cells following treatment with the receptor activator of nuclear factor-kappa-B ligand (RANKL). MATERIAL AND METHODS RAFLS cells were cultured in vitro and were divided into six study groups: a normal control group; a miR-515-5p mimic group; a miR-515-5p inhibitor group; a RANKL (50 ng/ml) treatment group; a miR-515-5p mimic+RANKL treatment group; and a miR-515-5p inhibitor+RANKL treatment group. The luciferase assay was used to determine the effects of miR-515-5p on the WISP1 expression. Cell proliferation, cell apoptosis, the cell cycle, and protein expression were determined using the Cell Counting Kit-8 (CCK-8) assay, flow cytometry, Western blot, and real-time polymerase chain reaction (RT-PCR). RESULTS The luciferase assay showed that the effects of miR-515-on the 3'-UTR of WISP1 inhibited the gene expression. The miR-515-5p mimics promoted cell proliferation, reduced apoptosis, and promoted the cell cycle. The miR-515-5p mimics reduced, the expression of TLR4, WISP1, and JNK at the mRNA level, while the miR-515-5p inhibitor promoted the expression of TLR4, WISP1, and JNK. Both the miR-515-5p inhibitor and mimic promoted the phosphorylation of AKT in RAFLS cells treated with or without RANKL compared with the control, and the miR-515-5p inhibitor promoted the phosphorylation of JNK in the RAFLS cells. CONCLUSIONS In RAFLS cells, miR-515-5p inhibited the expression of the WISP1 gene, and treatment with RANKL inhibited the TLR4/JNK signaling pathway.
Assuntos
Artrite Reumatoide/metabolismo , Proteínas de Sinalização Intercelular CCN/genética , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , MicroRNAs/metabolismo , Proteínas Proto-Oncogênicas/genética , Ligante RANK/farmacologia , Receptor 4 Toll-Like/metabolismo , Regiões 3' não Traduzidas , Apoptose/efeitos dos fármacos , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/genética , Artrite Reumatoide/patologia , Materiais Biomiméticos/farmacologia , Proteínas de Sinalização Intercelular CCN/antagonistas & inibidores , Proteínas de Sinalização Intercelular CCN/biossíntese , Proteínas de Sinalização Intercelular CCN/metabolismo , Técnicas de Cultura de Células , Proliferação de Células/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Expressão Gênica/efeitos dos fármacos , Células HEK293 , Humanos , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas/metabolismo , Ligante RANK/metabolismo , Transdução de Sinais , Sinoviócitos/metabolismo , Sinoviócitos/patologiaRESUMO
BACKGROUND: Accumulating evidence has shown the important roles of long non-coding RNAs (lncRNAs) in acute lung injury (ALI). This study aimed to investigate the potential role of lncRNA small nucleolar RNA host gene 14 (SNHG14) in lipopolysaccharides (LPS)-induced ALI. METHODS: Expression of SNHG14, microRNA-34c-3p (miR-34c-3p) and Wnt1 inducible signaling pathway protein 1 (WISP1) in LPS-exposed mouse alveolar macrophages (MH-S) and lung tissues from mice with LPS-induced ALI was determined by reverse transcription quantitative polymerase chain reaction. The interactions among SNHG14, miR-34c-3p and WISP1 were analyzed by dual-luciferase reporter and RIP assays. Using gain-of-function or loss-of-function approaches, the contents of proinflammatory proteins were determined and MH-S cell viability was assessed to evaluate the in vitro functions of SNHG14, miR-34c-3p and WISP1, and wet/dry weight ratio and proinflammatory proteins in lung tissues were determined to assess their in vivo effects. RESULTS: SNHG14 and WISP1 expression was increased, while miR-34c-3p was decreased in ALI models. SNHG14 bound to miR-34c-3p, resulting in impaired miR-34c-3p-dependent down-regulation of WISP1. Both SNHG14 silencing and miR-34c-3p over-expression reduced the levels of proinflammatory proteins IL-18, IL-1ß, TNF-α and IL-6 and inhibited MH-S cell viability. SNHG14 silencing or miR-34c-3p over-expression decreased the wet/dry weight ratio in lung tissues from ALI mice. The reductions induced by SNHG14 silencing or miR-34c-3p over-expression were rescued by WISP1 over-expression. CONCLUSION: This study demonstrated that lncRNA SNHG14 silencing alleviated inflammation in LPS-induced ALI through miR-34c-3p-mediated inhibition of WISP1. Our findings suggest that lncRNA SNHG14 may serve as a therapeutic target for ALI.
Assuntos
Lesão Pulmonar Aguda/metabolismo , Proteínas de Sinalização Intercelular CCN/biossíntese , Regulação para Baixo/fisiologia , Lipopolissacarídeos/toxicidade , MicroRNAs/biossíntese , Proteínas Proto-Oncogênicas/biossíntese , RNA Longo não Codificante/biossíntese , Lesão Pulmonar Aguda/induzido quimicamente , Animais , Proteínas de Sinalização Intercelular CCN/antagonistas & inibidores , Proteínas de Sinalização Intercelular CCN/genética , Células Cultivadas , Regulação para Baixo/efeitos dos fármacos , Macrófagos Alveolares/efeitos dos fármacos , Macrófagos Alveolares/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos ICR , MicroRNAs/genética , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/genética , RNA Longo não Codificante/antagonistas & inibidores , RNA Longo não Codificante/genética , Distribuição AleatóriaRESUMO
OBJECTIVE: The objective of this study was to determine the prognostic and predictive impact of ß-catenin, TCF21 and WISP1 expression in patients with squamous cell carcinomas of the head and neck who underwent primary radiotherapy or concomitant chemoradiotherapy. STUDY DESIGN: Prospective cohort study. SETTING: University hospital. PARTICIPANTS: Protein expression profiles of ß-catenin, TCF21, WISP1 and p16 were determined by immunohistochemical analyses in tissue samples of 59 untreated patients. Expression was correlated with different outcome parameters. MAIN OUTCOME MEASURES: Impact of TNM classification, grading, sex, age, gender, type of therapy, response to therapy and p16 status on disease-specific (DSS) and disease-free survival (DFS). RESULTS: Patients with high expression of TCF21 were associated with significantly worse disease-specific survival (P = 0.005). In a multivariable analysis, TCF21 was a significant determinant of disease-specific survival. (HR 3.01; P = 0.036). Conversely, low expression of ß-catenin (P = 0.025) and WISP1 (P = 0.037) revealed a better response to radiotherapy. CONCLUSION: Since data show that TCF21 is a prognostic factor for disease-specific survival and WISP1 and ß-catenin are predictive factors for clinical outcome after definitive radiotherapy, further studies are warranted to prove these preliminary but very promising findings.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/biossíntese , Proteínas de Sinalização Intercelular CCN/biossíntese , Neoplasias de Cabeça e Pescoço/metabolismo , Estadiamento de Neoplasias , Proteínas Proto-Oncogênicas/biossíntese , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , beta Catenina/biossíntese , Adulto , Áustria/epidemiologia , Biomarcadores Tumorais/biossíntese , Quimiorradioterapia , Feminino , Seguimentos , Neoplasias de Cabeça e Pescoço/mortalidade , Neoplasias de Cabeça e Pescoço/terapia , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Prognóstico , Estudos Prospectivos , Carcinoma de Células Escamosas de Cabeça e Pescoço/mortalidade , Carcinoma de Células Escamosas de Cabeça e Pescoço/terapia , Taxa de Sobrevida/tendênciasRESUMO
Wnt1-inducible signaling pathway protein-1 is a cysteine-rich protein that belongs to the CCN family, which has been implicated in mediating the occurrence and progression through distinct molecular mechanisms in several tumor types. However, the association of Wnt1-inducible signaling pathway protein-1 with gastric cancer and the related molecular mechanisms remain to be elucidated. Therefore, this study aimed to clarify the biological role of Wnt1-inducible signaling pathway protein-1 in the proliferation, migration, and invasion in gastric cancer cells and further investigated the associated molecular mechanism on these biological functions. We first detected the expression level of Wnt1-inducible signaling pathway protein-1 in gastric cancer, and the reverse transcription polymerase chain reaction have shown that Wnt1-inducible signaling pathway protein-1 expression levels were upregulated in gastric cancer tissues. The expression of Wnt1-inducible signaling pathway protein-1 in gastric cancer cell lines was also detected by quantitative real-time polymerase chain reaction and Western blotting. Furthermore, two gastric cancer cell lines with high expression of Wnt1-inducible signaling pathway protein-1 were selected to explore the biological function of Wnt1-inducible signaling pathway protein-1 in gastric cancer. Function assays indicated that knockdown of Wnt1-inducible signaling pathway protein-1 suppressed cell proliferation, migration, and invasion in BGC-823 and AGS gastric cancer cells. Further investigation of mechanisms suggested that cyclinD1 was identified as one of Wnt1-inducible signaling pathway protein-1 related genes to accelerate proliferation in gastric cancer cells. In addition, one pathway of Wnt1-inducible signaling pathway protein-1 induced migration and invasion was mainly through the enhancement of epithelial-to-mesenchymal transition progression. Taken together, our findings presented the first evidence that Wnt1-inducible signaling pathway protein-1 was upregulated in gastric cancer and acted as an oncogene by promoting proliferation, migration, and invasion in gastric cancer cells.
Assuntos
Biomarcadores Tumorais/biossíntese , Proteínas de Sinalização Intercelular CCN/biossíntese , Proliferação de Células/genética , Proteínas Proto-Oncogênicas/biossíntese , Neoplasias Gástricas/genética , Biomarcadores Tumorais/genética , Proteínas de Sinalização Intercelular CCN/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Transição Epitelial-Mesenquimal/genética , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Proteínas Proto-Oncogênicas/genética , Transdução de Sinais , Neoplasias Gástricas/patologiaRESUMO
OBJECTIVE: Osteoarthritis (OA) and rheumatoid arthritis (RA) are widespread disabling joint disorders that are considered to be polygenic in nature. This study investigated the spatial expression patterns of all six known human CCN genes using end-stage OA and RA joint samples. DESIGN: We performed in situ hybridization and histological analysis to investigate the spatial expression patterns of human CCN genes using joint tissues obtained during total knee and hip joint replacement procedures on patients with advanced OA or RA. Normal joint tissues taken while performing bipolar hip replacement surgeries were used as controls. RESULTS: All CCN genes were expressed at higher levels in OA and RA synovial samples as compared with normal controls. Whereas CCN3 and CCN6 were undetectable in control, OA, and RA cartilage, CCN1, CCN2, CCN4, and CCN5 were expressed to a greater extent in OA and RA knee cartilage. CONCLUSIONS: Our results indicate an involvement of several CCN genes in the pathophysiology of OA and RA.
Assuntos
Artrite Reumatoide/genética , Proteínas de Sinalização Intercelular CCN/genética , Cartilagem Articular/metabolismo , Regulação da Expressão Gênica , Osteoartrite/genética , RNA Mensageiro/genética , Membrana Sinovial/metabolismo , Idoso , Idoso de 80 Anos ou mais , Artrite Reumatoide/metabolismo , Proteínas de Sinalização Intercelular CCN/biossíntese , Feminino , Perfilação da Expressão Gênica/métodos , Humanos , Hibridização In Situ , Masculino , Osteoartrite/metabolismo , Reação em Cadeia da PolimeraseRESUMO
The CCN proteins are extracellular matrix associated proteins involved in critical cell activities and several aggressive forms of cancer. The proteins share a modular structure of four discrete domains and 38 conserved cysteine residues. The absence of any structural information of these proteins has resulted in a need for the ability to produce substantial amounts of pure CCN protein. Through bacterial expression and inclusion body based purification, pure recombinant CCN proteins have been produced for use in structural and biochemical experiments.
Assuntos
Proteínas de Sinalização Intercelular CCN/biossíntese , Proteínas de Sinalização Intercelular CCN/isolamento & purificação , Escherichia coli/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas de Sinalização Intercelular CCN/química , Clonagem Molecular , Escherichia coli/química , Escherichia coli/genética , Humanos , Dobramento de Proteína , Proteínas Recombinantes/químicaRESUMO
BACKGROUND: Breast cancer (BRCA) is the most prevalent cancer that threatens female health. A growing body of evidence has demonstrated the non-negligible effects of messenger RNAs (mRNAs) on biological processes involved in cancers; however, there is no definite conclusion regarding the role of mRNAs in predicting the prognosis of BRCA patients. MATERIALS AND METHODS: We systematically screened the mRNA expression landscape and clinical data of samples from the Cancer Genome Atlas (TCGA). Univariate Cox analysis and robust likelihood-based survival analysis were conducted to identify key mRNAs associated with BRCA. Furthermore, risk scores based on multivariate Cox analysis divided the training set into high-risk and low-risk groups. ROC analysis determined the optimal cut-off point for patient classification of risk levels. The prognostic model was additionally validated in the testing set and complete dataset. Finally, we plotted the survival curves for the mRNAs used in our model. RESULTS: We obtained the original expression data of 13,617 mRNAs from a total of 1088 samples. After comprehensive survival analysis, the four-mRNA (ACSL1, OTUD3, PKD1L2, and WISP1) prognosis risk assessment model was constructed. Furthermore, the area under cure (AUC) was 0.834, indicating that the model was meaningful and reasonable. In each dataset, analysis based on the four-mRNA signature risk score indicated that the survival status of the group with high risk score was worse than that of the group with low risk scores. Patients with strong mRNA expression of OTUD3, PKD1L2, and WISP1 tended to have good prognosis, whereas patients with high ACSL1 expression tended to have poor prognosis. CONCLUSION: In summary, we constructed a four-mRNA prognosis risk assessment model for BRCA. The newly developed model offers more possibilities for assessing prognosis and guiding the selection of better treatment strategies for BRCA.
Assuntos
Neoplasias da Mama , Bases de Dados de Ácidos Nucleicos , Modelos Biológicos , RNA Mensageiro/biossíntese , RNA Neoplásico/biossíntese , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Proteínas de Sinalização Intercelular CCN/biossíntese , Proteínas de Sinalização Intercelular CCN/genética , Coenzima A Ligases/biossíntese , Coenzima A Ligases/genética , Intervalo Livre de Doença , Feminino , Humanos , Valor Preditivo dos Testes , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas/genética , RNA Mensageiro/genética , RNA Neoplásico/genética , Receptores Acoplados a Proteínas G/biossíntese , Receptores Acoplados a Proteínas G/genética , Taxa de Sobrevida , Proteases Específicas de Ubiquitina/biossíntese , Proteases Específicas de Ubiquitina/genéticaRESUMO
Brevibacillus choshinensis is an excellent host for the production of secretory proteins. This host has also been applied successfully to efficient production of CCN proteins. Described herein are methods of constructing plasmids for CCN protein production (IGFBP-, VWC-, TSP-, and CT-domain) with Brevibacillus as a host, cultivation methods for protein production, and methods of purification for domain proteins using his-tag.
Assuntos
Brevibacillus/genética , Brevibacillus/metabolismo , Proteínas de Sinalização Intercelular CCN/biossíntese , Proteínas de Sinalização Intercelular CCN/genética , Proteínas Recombinantes de Fusão , Proteínas de Sinalização Intercelular CCN/isolamento & purificação , Clonagem Molecular , Diálise , Ordem dos Genes , Plasmídeos/genética , UltrafiltraçãoRESUMO
Recombinant CCN2 protein (rCCN2) is available from many companies; however, most of them are produced in E. coli. To investigate true functions of rCCN2, glycosylated protein with proper folding needs to be used. Therefore, we use rCCN2 produced by mammalian cells. Conditioned medium (CM) of HeLa cells stably transfected with a CCN2 expression vector are collected, and the recombinant CCN2 protein produced and secreted into the CM is purified by two-step chromatography, first with a heparin affinity column and then with an anti-CCN2 affinity column prepared with a monoclonal antibody against CCN2. The purified rCCN2 shows the bands of 36-38 kDa with sliver staining after gel electrophoresis, which can be confirmed by Western blotting. This chapter describes these methods in detail.
Assuntos
Proteínas de Sinalização Intercelular CCN/biossíntese , Proteínas de Sinalização Intercelular CCN/genética , Células HeLa , Proteínas Recombinantes de Fusão , Proteínas de Sinalização Intercelular CCN/isolamento & purificação , Cromatografia de Afinidade/métodos , Expressão Gênica , Ordem dos Genes , Humanos , Plasmídeos/genética , Transfecção/métodosRESUMO
Recombinant proteins are important tools for understanding molecular functions in vitro. Recent progress in the generation of recombinant proteins is amazing. However, when we plan to produce them, we should choose the best method according to the nature and the use of the target recombinant protein. Degradation and mis-folding are major problems in producing active recombinant CCN2. The method shown in this chapter describes the appropriate conditions under which we can produce CCN2 and its truncated fragments in Escherichia coli.
Assuntos
Proteínas de Sinalização Intercelular CCN/biossíntese , Proteínas de Sinalização Intercelular CCN/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Recombinantes de Fusão , Proteínas de Sinalização Intercelular CCN/isolamento & purificação , Cromatografia de Afinidade/métodos , DNA Complementar , Expressão Gênica , Ordem dos Genes , Vetores Genéticos/genética , HumanosRESUMO
Wnt-induced secreted protein-1 (WISP1) is an extracellular matrix protein that has been reported in cancer researches. Our previous studies on WISP1 implied it could be a harmful mediator in septic mice. However, its role in liver ischemia reperfusion (I/R) injury is unknown. This study investigated the effects of WISP1 on liver I/R damage. Male C57BL/6 wild-type mice were used to undergo 60 min segmental (70%) ischemia. WISP1 expression was measured after indicated time points of reperfusion. Anti-WISP1 antibody was injected intraperitoneally to mice. Toll-like receptor 4 (TLR4) knockout mice and TIR-domain-containing adaptor inducing interferon-ß (TRIF) knockout mice were adopted in this study. WISP1 was significantly enhanced after 6 h of reperfusion when compared with sham treated mice and significantly decreased either by TLR4 knockout mice or TRIF knockout mice. Anti-WISP1 antibody significantly decreased serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), pathological changes and pro-inflammatory cytokine levels in the mice following I/R. Furthermore, significantly increased serum transaminase levels were found in C57 wild-type mice treated with recombinant WISP1 protein, but not found in TLR4 knockout or TRIF knockout mice subjected to liver I/R. Taken together, WISP1 might contribute to hepatic ischemia reperfusion injury in mice and possibly depends on TLR4/TRIF signaling.
Assuntos
Proteínas de Sinalização Intercelular CCN/biossíntese , Regulação da Expressão Gênica , Fígado/metabolismo , Proteínas Proto-Oncogênicas/biossíntese , Traumatismo por Reperfusão/metabolismo , Transdução de Sinais , Receptor 4 Toll-Like/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Proteínas de Sinalização Intercelular CCN/genética , Fígado/patologia , Masculino , Camundongos , Camundongos Knockout , Proteínas Proto-Oncogênicas/genética , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/patologia , Receptor 4 Toll-Like/genéticaRESUMO
BACKGROUND: Previous work characterized variants of the EL4 murine lymphoma cell line. Some are non-metastatic, and others metastatic, in syngenic mice. In addition, metastatic EL4 cells were stably transfected with phospholipase D2 (PLD2), which further enhanced metastasis. MATERIALS AND METHODS: Microarray analyses of mRNA expression was performed for non-metastatic, metastatic, and PLD2-expressing metastatic EL4 cells. RESULTS: Many differences were observed between non-metastatic and metastatic cell lines. One of the most striking new findings was up-regulation of mRNA for the matricellular protein WNT1-inducible signaling pathway protein 1 (CCN4) in metastatic cells; increased protein expression was verified by immunoblotting and immunocytochemistry. Other differentially expressed genes included those for reproductive homeobox 5 (Rhox5; increased in metastatic) and cystatin 7 (Cst7; decreased in metastatic). Differences between PLD2-expressing and parental cell lines were limited but included the signaling proteins Ras guanyl releasing protein 1 (RGS18; increased with PLD2) and suppressor of cytokine signaling 2 (SOCS2; decreased with PLD2). CONCLUSION: The results provide insights into signaling pathways potentially involved in conferring metastatic ability on lymphoma cells.
Assuntos
Proteínas de Sinalização Intercelular CCN/biossíntese , Regulação Neoplásica da Expressão Gênica/genética , Linfoma/genética , Análise em Microsséries , Proteínas Proto-Oncogênicas/biossíntese , Animais , Proteínas de Sinalização Intercelular CCN/genética , Linhagem Celular Tumoral , Cistatinas/biossíntese , Cistatinas/genética , Modelos Animais de Doenças , Proteínas de Homeodomínio/biossíntese , Proteínas de Homeodomínio/genética , Humanos , Linfoma/patologia , Camundongos , Metástase Neoplásica , Fosfolipase D/biossíntese , Fosfolipase D/genética , Proteínas Proto-Oncogênicas/genética , RNA Mensageiro/biossíntese , Transdução de Sinais/genética , Proteínas Supressoras da Sinalização de Citocina/biossíntese , Proteínas Supressoras da Sinalização de Citocina/genética , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genéticaRESUMO
BACKGROUND/AIM: Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) insufficiency is commonly found in breast cancer patients with metastasis. We investigated the mechanisms by which PTEN affects breast cancer metastatic behavior. MATERIALS AND METHODS: Migration and invasion assay, western blot, immunofluorescent staining and zebrafish animal model were applied. RESULTS: We showed that PTEN insufficiency induced an increase in MCF-7 cell migration and invasion through induction of epithelial-mesenchymal transition (EMT), which was triggered by up-regulation of the EMT-inducing transcriptional factors Zeb1, Zeb2, Snail, Slug and Twist. Simultaneously, E-cadherin expression was inhibited and P-cadherin was up-regulated. Further, WNT1 inducible signaling pathway protein 1 (WISP1) and lipocalin-2 (LCN2) expressions were increased after PTEN knockdown in MCF-7 cells, which also exhibited increased filamentous actin (F-actin) synthesis and extracellular matrix metalloproteinase-2 (MMP-2) and MMP-9 expression. We further showed that PTEN knockdown in MCF-7 cells could increase cell migration in the xenograft zebrafish model. CONCLUSION: Our findings reveal new therapeutic targets for breast cancer patients with PTEN insufficiency.
Assuntos
Neoplasias da Mama/genética , Transição Epitelial-Mesenquimal/genética , Invasividade Neoplásica/genética , PTEN Fosfo-Hidrolase/genética , Animais , Neoplasias da Mama/patologia , Proteínas de Sinalização Intercelular CCN/biossíntese , Movimento Celular/genética , Modelos Animais de Doenças , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Proteínas de Homeodomínio/biossíntese , Humanos , Células MCF-7 , Metaloproteinase 2 da Matriz/biossíntese , Metaloproteinase 9 da Matriz/biossíntese , Metástase Neoplásica , PTEN Fosfo-Hidrolase/biossíntese , Proteínas Repressoras/biossíntese , Fatores de Transcrição da Família Snail/biossíntese , Proteína 1 Relacionada a Twist/biossíntese , Proteína Wnt1/biossíntese , Ensaios Antitumorais Modelo de Xenoenxerto , Peixe-Zebra , Proteínas de Peixe-Zebra/biossíntese , Homeobox 2 de Ligação a E-box com Dedos de Zinco , Homeobox 1 de Ligação a E-box em Dedo de Zinco/biossínteseRESUMO
Studies have reported that the CCN family of proteins plays an important role in stimulating tumorigenesis. However, the relationship between the CCN protein family members and the features of hepatocellular carcinoma (HCC) remains unclear. The objective of this study was to determine the relationship between the expression levels of CCN protein family members and the features of HCC. Expression levels of the CCN family of proteins in 80-paired primary HCC samples and 11 normal liver samples were determined by a quantitative real-time PCR assay. Enhanced expression of nephroblastoma overexpressed protein (NOV) and decreased expression of Wnt-induced secreted protein 1 (WISP1), cysteine-rich protein 61 (CYR61) and connective tissue growth factor (CTGF) were found in HCC samples when compared to levels in matched non-cancerous tissues. No significant difference in WISP2 was found between matched-pair samples; only a few samples showed WISP3 expression. Furthermore, the expression levels of NOV, WISP1 and CYR61 were closely correlated with certain clinical features, including venous invasion, cellular differentiation, pTNM stage, disease-free survival and overall survival. Our results suggest that HCC progression may be enhanced by NOV and suppressed by WISP1 and CYR61. Our statistical analysis suggests that these proteins may be valuable in determining the prognosis of this deadly disease and directs attention to modulating the levels of these proteins as a potential mode of therapy.
Assuntos
Proteínas de Sinalização Intercelular CCN/biossíntese , Carcinoma Hepatocelular/patologia , Transformação Celular Neoplásica/patologia , Neoplasias Hepáticas/patologia , Fator de Crescimento do Tecido Conjuntivo/biossíntese , Proteína Rica em Cisteína 61/biossíntese , Progressão da Doença , Intervalo Livre de Doença , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteína Sobre-Expressa em Nefroblastoma/biossíntese , Reação em Cadeia da Polimerase , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Repressoras/biossínteseRESUMO
Radiotherapy is a primary treatment modality for esophageal squamous cell carcinoma (ESCC). However, most of patients benefited little from radiotherapy due to refractory radioresistance. We found that WISP1, a downstream target gene of Wnt/ß-catenin pathway, was re-expressed in 67.3% of ESCC patients as an oncofetal gene. Expression of WISP1 predicted prognosis of ESCC patients treated with radiotherapy. Overall survival in WISP1-positive patients was significantly poorer than in WISP1-negative patients. Serum concentration of WISP1 after radiotherapy reversely correlated with relapse-free survival. Gain and loss of function studies confirmed that WISP1 mediated radioresistance both in esophageal squamous cancer cells and in xenograft tumor models. Further studies revealed that WISP1 contributed to radioresistance primarily by repressing irradiation-induced DNA damage and activating PI3K kinase. LncRNA BOKAS was up-regulated following radiation and promoted WISP1 expression and resultant radioresistance. Furthermore, WISP1 facilitated its own expression in response to radiation, creating a positive feedback loop and increased radioresistance. Our study revealed WISP1 as a potential target to overcome radioresistance in ESCC.
Assuntos
Proteínas de Sinalização Intercelular CCN/biossíntese , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/radioterapia , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/radioterapia , Proteínas Proto-Oncogênicas/biossíntese , Animais , Proteínas de Sinalização Intercelular CCN/sangue , Proteínas de Sinalização Intercelular CCN/genética , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Dano ao DNA , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago , Feminino , Xenoenxertos , Humanos , Camundongos Endogâmicos BALB C , Camundongos Nus , Fosfatidilinositol 3-Quinases/metabolismo , Prognóstico , Proteínas Proto-Oncogênicas/sangue , Proteínas Proto-Oncogênicas/genética , Tolerância a Radiação , Transfecção , Via de Sinalização WntRESUMO
Epidural ï¬brosis is characterized by the development of dense and thick scar tissue adjacent to the dural mater and ranked as the major contributor for post-operative pain recurrence after laminectomy or discectomy. Recently, CCN5 exhibited an inhibitory effect on connective tissue growth factor (CTGF)/CCN2 (a critical regulator for fibrotic disease)mediated fibrogenesis. However, its function in epidural fibrosis and the underlying mechanisms involved remain to be determined. In this study, an obvious downregulation of CCN5 was observed in scar tissues from laminectomized rats, concomitant with a marked upregulation of CCN2, suggesting a potential negative regulatory role of CCN5 in fibrogenesis. Furthermore, CCN5 overexpression notably mitigated transforming growth factorß1-enhanced fibroblast viability and proliferation. Of note, CCN5 upregulation inhibited the switch of fibroblasts into myofibroblasts as its overexpression abrogated the expression of the myoï¬broblast marker, α-smooth muscle actin (α-SMA). CCN5 upregulation also reduced an increase in collagen type I, α1 (COL1A1) and total collagen concentrations. Additionally, CCN5 over-expression decreased CCN2 expression and increased Smad6 phosphorylation. Mechanism analysis revealed that blocking Smad6 signaling significantly ameliorated the inhibitory effect of CCN5 on the CCN2 levels, accompanied by the reduction in cell proliferation and collagen production. These results confirm that CCN5 exerts an anti-fibrotic function by regulating the Smad6-CCN2 pathway, thereby indicating a potential approach for ameliorating epidural ï¬brosis after laminectomy.
Assuntos
Proteínas de Sinalização Intercelular CCN/metabolismo , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Fibrose/patologia , Proteínas Repressoras/metabolismo , Proteína Smad6/metabolismo , Coluna Vertebral/patologia , Actinas/biossíntese , Animais , Proteínas de Sinalização Intercelular CCN/biossíntese , Proliferação de Células , Células Cultivadas , Colágeno Tipo I/biossíntese , Cadeia alfa 1 do Colágeno Tipo I , Fator de Crescimento do Tecido Conjuntivo/biossíntese , Fibroblastos/fisiologia , Laminectomia/efeitos adversos , Masculino , Fosforilação , Interferência de RNA , RNA Interferente Pequeno , Ratos , Ratos Endogâmicos Lew , Proteínas Repressoras/biossíntese , Transdução de Sinais , Proteína Smad6/genética , Fator de Crescimento Transformador beta1/metabolismoRESUMO
The CCN family of proteins is composed of six extracellular matrix-associated proteins that play crucial roles in skeletal development, wound healing, fibrosis, and cancer. Members of the CCN family share four conserved cysteine-rich modular domains that trigger signal transduction in cell adhesion, migration, proliferation, differentiation, and survival through direct binding to specific integrin receptors and heparan sulfate proteoglycans. In the present review, we discuss the roles of the CCN family proteins in regulating resident cells of the bone microenvironment. In vertebrate development, the CCN family plays a critical role in osteo/chondrogenesis and vasculo/angiogenesis. These effects are regulated through signaling via integrins, bone morphogenetic protein, vascular endothelial growth factor, Wnt, and Notch via direct binding to CCN family proteins. Due to the important roles of CCN family proteins in skeletal development, abnormal expression of CCN proteins is related to the tumorigenesis of primary bone tumors such as osteosarcoma, Ewing sarcoma, and chondrosarcoma. Additionally, emerging studies have suggested that CCN proteins may affect progression of secondary metastatic bone tumors by moderating the bone microenvironment. CCN proteins could therefore serve as potential therapeutic targets for drug development against primary and metastatic bone tumors.
Assuntos
Neoplasias Ósseas/genética , Proteínas de Sinalização Intercelular CCN/genética , Diferenciação Celular/genética , Terapia de Alvo Molecular , Neovascularização Patológica/genética , Desenvolvimento Ósseo/genética , Neoplasias Ósseas/patologia , Neoplasias Ósseas/terapia , Proteínas de Sinalização Intercelular CCN/biossíntese , Adesão Celular/genética , Humanos , Transdução de Sinais/genéticaRESUMO
Idiopathic pulmonary fibrosis (IPF) is the most common and fatal form of idiopathic interstitial pneumonia. MicroRNAs (miRNAs), short, single-stranded RNAs that regulate protein expression in a post-transcriptional manner, have recently been demonstrated to contribute to IPF pathogenesis. We have previously identified WNT1-inducible signaling pathway protein 1 (WISP1) as a highly expressed pro-fibrotic mediator in IPF, but the underlying mechanisms resulting in increased WISP1 expression, remain elusive. Here, we investigated whether WISP1 is a target of miRNA regulation. We applied a novel supervised machine learning approach, which predicted miR-30a/d and miR-92a target sites in regions of the human WISP1 3'UTR preferentially bound by the miRNA ribonucleoprotein complex. Both miRNAs were decreased in IPF samples, whereas WISP1 protein was increased. We demonstrated further that transforming growth factor (TGF)-ß1-induced WISP1 expression in primary lung fibroblasts in vitro and lung homogenates in vivo. Notably, miR-30a and miR-92a reversed TGF-ß1-induced WISP1 mRNA expression in lung fibroblasts. Moreover, miR-92a inhibition increased WISP1 protein expression in lung fibroblasts. An inverse relationship for WISP1 and miR-92a was found in a TGF-ß1 dependent lung fibrosis model in vivo. Finally, we found significantly increased WISP1 expression in primary IPF fibroblasts, which negatively correlated with miR-92a level ex vivo. Altogether, our findings indicate a regulatory role of miR-92a for WISP1 expression in pulmonary fibrosis.
Assuntos
Proteínas de Sinalização Intercelular CCN/biossíntese , Fibrose Pulmonar Idiopática/genética , MicroRNAs/biossíntese , Proteínas Proto-Oncogênicas/biossíntese , Fator de Crescimento Transformador beta1/genética , Animais , Inteligência Artificial , Regulação da Expressão Gênica , Humanos , Fibrose Pulmonar Idiopática/patologia , MicroRNAs/antagonistas & inibidores , Ratos , Transdução de Sinais , Fator de Crescimento Transformador beta1/metabolismoRESUMO
Aggressive fibromatosis (AF) represents a group of tumors with a variable and unpredictable clinical course, characterized by a monoclonal proliferation of myofibroblastic cells. The optimal treatment for AF remains unclear. Identification and validation of genes whose expression patterns are associated with AF may elucidate biological mechanisms in AF, and aid treatment selection. This study was designed to examine the protein expression by immunohistochemistry (IHC) of four genes, ADAM12, FAP, SOX11, and WISP1, that were found in an earlier study to be uniquely overexpressed in AF compared with normal tissues. Digital image analysis was performed to evaluate inter- and intratumor heterogeneity, and correlate protein expression with histologic features, including a histopathologic assessment of tumor activity, defined by nuclear chromatin density ratio (CDR). AF tumors exhibited marked inter- and intratumor histologic heterogeneity. Pathologic assessment of tumor activity and digital assessment of average nuclear size and CDR were all significantly correlated. IHC revealed protein expression of all four genes. IHC staining for ADAM12, FAP, and WISP1 correlated with CDR and was higher, whereas SOX11 staining was lower in tumors with earlier recurrence following excision. All four proteins were expressed, and the regional variation in tumor activity within and among AF cases was demonstrated. A spatial correlation between protein expression and nuclear morphology was observed. IHC also correlated with the probability of recurrence following excision. These proteins may be involved in AF pathogenesis and the corresponding pathways could serve as potential targets of therapy.
Assuntos
Proteínas ADAM/biossíntese , Proteínas de Sinalização Intercelular CCN/biossíntese , Fibromatose Agressiva/genética , Gelatinases/biossíntese , Proteínas de Membrana/biossíntese , Proteínas Proto-Oncogênicas/biossíntese , Fatores de Transcrição SOXC/biossíntese , Serina Endopeptidases/biossíntese , Proteínas ADAM/genética , Proteína ADAM12 , Proteínas de Sinalização Intercelular CCN/genética , Cromatina/química , Cromatina/genética , Endopeptidases , Fibromatose Agressiva/patologia , Gelatinases/genética , Regulação Neoplásica da Expressão Gênica/genética , Heterogeneidade Genética , Humanos , Proteínas de Membrana/genética , Proteínas Proto-Oncogênicas/genética , Fatores de Transcrição SOXC/genética , Serina Endopeptidases/genéticaRESUMO
Individuals with the autosomal recessive skeletal disorder Progressive Pseudorheumatoid Dysplasia have loss-of-function mutations in WISP3, and aberrant WISP3 expression has been detected in tumors from patients with colon and breast cancer. In mice however, neither absence nor over-expression of WISP3 was found to cause a phenotype, and endogenous Wisp3 expression has been difficult to detect. To confirm that Wisp3 knockout mice have no phenotype and to identify potential sites of endogenous Wisp3 expression, we generated mice with a knockin allele (Wisp3 (GFP-Cre)) designed to express Green Fluorescent Protein (GFP) and Cre-recombinase instead of WISP3. Heterozygous and homozygous knockin mice were fertile and indistinguishable from their wild-type littermates, confirming that mice lacking Wisp3 have no phenotype. We could not detect GFP-expression from the knockin allele, but we could detect Cre-expression after crossing mice with the knockin allele to Cre-reporter mice; the double heterozygous offspring had evidence of Cre-mediated recombination in several tissues. The only tissue that had high levels of Cre-mediated recombination was the testis, where recombination in spermatocytes occurred by early prophase of meiosis I. As a consequence, males that were double heterozygous for a Wisp3 (GFP-Cre) and a floxed allele only contributed a recombined allele to their offspring. We detected no evidence of Cre-mediated recombination in the female ovary, although when double heterozygous females contributed the reporter allele to their offspring it had recombined ~7% of the time. Wisp3 (GFP-Cre) expression therefore occurs less frequently and most likely at a later stage of oocyte development in female mice compared to male mice. We conclude that although WISP3 is dispensable in mice, male mice with a Wisp3 (GFP-Cre) allele (Jackson Laboratory stock # 017685) will be useful for studying early prophase of meiosis I and for efficiently recombining floxed alleles that are passed to offspring.