RESUMO
BACKGROUND: Hepatitis B virus (HBV) infection is a major concern regarding blood safety in countries with a high HBV prevalence, such as China. We aimed to understand the prevalence of HBV infection among blood donors in Chongqing and provide an important basis for developing appropriate blood screening strategies. METHODS: Dual enzyme-linked immunosorbent assays (ELISAs) for hepatitis B surface antigen (HBsAg) were conducted in parallel with nucleic acid testing (NAT) of donors. All HBsAg-reactive and/or HBV DNA-positive blood samples were tested for HBsAg and hepatitis B DNA levels. RESULTS: A total of 117,927 blood donor samples were collected from the Chongqing Blood Center between April 2020 and November 2020. In total, 473 HBV-ineligible samples were retained for HBsAg and DNA confirmation. A total of 272 samples were confirmed to be HBsAg+, including 2 HBV DNA - and 270 HBV DNA + samples. A total of 201 donations were HBsAg-, including 72 HBV DNA - samples. The rate of HBV infection was 65.33% (309/473) in men, which was significantly higher than that in women (p < 0.001). The HBV failure rate was higher among the first-time donors (p < 0.05). Of the 182 NAT R/HBsAg N/N samples (Nucleic acid test reactivity/2 anti-HBsAg tests negative), 37.91% (69/182) were false positives. The proportion of hepatitis B infections in the 18 NAT R/HBsAg N/R (Nucleic acid test reactivity/1 anti-HBsAg tests negative) samples was 94.44% (17/18), of which 50% (9/18) were occult HBV infection. A total of 95.83% (69/72) of the false positives were from the NAT R/HBsAg N/N group, and 58.33% (42/72) were first-time donors. CONCLUSION: Our data showed a strikingly high HBV infection rate among blood donors in Chongqing. Double ELISA and single NAT can effectively prevent HBV leakage and improve blood safety. First-time donors have a high rate of HBV transplant failure; therefore, donors should be retained and recruited from low-risk groups.
Assuntos
Doadores de Sangue , DNA Viral , Ensaio de Imunoadsorção Enzimática , Antígenos de Superfície da Hepatite B , Vírus da Hepatite B , Hepatite B , Humanos , Doadores de Sangue/estatística & dados numéricos , China/epidemiologia , Feminino , Masculino , Hepatite B/epidemiologia , Hepatite B/diagnóstico , Hepatite B/sangue , Vírus da Hepatite B/genética , Vírus da Hepatite B/isolamento & purificação , Vírus da Hepatite B/imunologia , Antígenos de Superfície da Hepatite B/sangue , Adulto , DNA Viral/sangue , Pessoa de Meia-Idade , Prevalência , Adulto Jovem , AdolescenteRESUMO
Hypoxia is a key feature of tumor microenvironment that contributes to the development of breast cancer stem cells (BCSCs) with strong self-renewal properties. However, the specific mechanism underlying hypoxia in BCSC induction is not completely understood. Herein, we provide evidence that a novel hypoxia-specific circSTT3A is significantly upregulated in clinical breast cancer (BC) tissues, and is closely related to the clinical stage and poor prognosis of patients with BC. The study revealed that hypoxia-inducible factor 1 alpha (HIF1α)-regulated circSTT3A has a remarkable effect on mammosphere formation in breast cancer cells. Mechanistically, circSTT3A directly interacts with nucleotide-binding domain of heat shock protein 70 (HSP70), thereby facilitating the recruitment of phosphoglycerate kinase 1 (PGK1) via its substrate-binding domain, which reduces the ubiquitination and increases the stability of PGK1. The enhanced levels of PGK1 catalyze 1,3-diphosphoglycerate (1,3-BPG) into 3-phosphoglycerate (3-PG) leading to 3-PG accumulation and increased serine synthesis, S-adenosylmethionine (SAM) accumulation, and trimethylation of histone H3 lysine 4 (H3K4me3). The activation of the H3K4me3 contributes to BCSCs by increasing the transcriptional level of stemness-related factors. Especially, our work reveals that either loss of circSTT3A or PGK1 substantially suppresses tumor initiation and tumor growth, which dramatically increases the sensitivity of tumors to doxorubicin (DOX) in mice. Injection of PGK1-silenced spheroids with 3-PG can significantly reverse tumor initiation and growth in mice, thereby increasing tumor resistance to DOX. In conclusion, our study sheds light on the functional role of hypoxia in the maintenance of BCSCs via circSTT3A/HSP70/PGK1-mediated serine synthesis, which provides new insights into metabolic reprogramming, tumor initiation and growth. Our findings suggest that targeting circSTT3A alone or in combination with chemotherapy has potential clinical value for BC management.
Assuntos
Neoplasias da Mama , Humanos , Camundongos , Animais , Feminino , Neoplasias da Mama/metabolismo , Histonas/metabolismo , Hipóxia/metabolismo , Transformação Celular Neoplásica/metabolismo , Células-Tronco Neoplásicas/patologia , Linhagem Celular Tumoral , Microambiente TumoralRESUMO
Cancer-associated fibroblasts (CAFs) are the predominant stromal cells in the microenvironment and play important roles in tumor progression, including chemoresistance. However, the response of CAFs to chemotherapeutics and their effects on chemotherapeutic outcomes are largely unknown. In this study, we showed that epirubicin (EPI) treatment triggered ROS which initiated autophagy in CAFs, TCF12 inhibited autophagy flux and further promoted exosome secretion. Inhibition of EPI-induced reactive oxygen species (ROS) production with N-acetyl-L-cysteine (NAC) or suppression of autophagic initiation with short interfering RNA (siRNA) against ATG5 blunted exosome release from CAFs. Furthermore, exosome secreted from EPI-treated CAFs not only prevented ROS accumulation in CAFs but also upregulated the CXCR4 and c-Myc protein levels in recipient ER+ breast cancer cells, thus promoting EPI resistance of tumor cells. Together, the current study provides novel insights into the role of stressed CAFs in promoting tumor chemoresistance and reveal a new function of TCF12 in regulating autophagy impairment and exosome release.
Assuntos
Neoplasias da Mama , Fibroblastos Associados a Câncer , Exossomos , Humanos , Feminino , Fibroblastos Associados a Câncer/patologia , Neoplasias da Mama/patologia , Epirubicina/farmacologia , Epirubicina/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Resistencia a Medicamentos Antineoplásicos , Fibroblastos/metabolismo , Exossomos/metabolismo , Microambiente Tumoral , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismoRESUMO
This study aimed to investigate the prevalence of and risk factors for multidrug-resistant organism (MDRO) infection in the rehabilitation ward of a general hospital in Southwest China. We analyzed rehabilitation patients with nosocomial infections caused by MDROs from June 2016 to June 2020. MDRO infection pathogens and associated antibiotic resistance were calculated. Possible risk factors for MDRO-related infection in the neurorehabilitation ward were analyzed using chi-square, and logistic regression. A total of 112 strains of MDRO were found positive from 96 patients. The MDRO test-positive rate was 16.70% (96/575). Ninety-five MDRO strains were detected in sputum, of which 84.82% (95/112) were gram-negative bacteria. Acinetobacter baumannii (A. Baumannii), Pseudomonas aeruginosa (P. aeruginosa), and Klebsiella pneumonia (K. pneumonia) were the most frequently isolated MDRO strains. The logistic regression model and multifactorial analysis showed that long-term (≥ 7 days) antibiotic use (OR 6.901), history of tracheotomy (OR 4.458), and a low albumin level (< 40 g/L) (OR 2.749) were independent risk factors for the development of MDRO infection in patients in the rehabilitation ward (all P < 0.05). Gram-negative MRDOs were dominant in rehabilitation ward patients. Low albumin, history of a tracheostomy, and long-term use of antibiotics were independent risk factors for MRDO infection and are worthy of attention.
Assuntos
Farmacorresistência Bacteriana Múltipla , Reabilitação Neurológica , Albuminas , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Enterococcus , Bactérias Gram-Negativas , Hospitais Gerais , Humanos , Pseudomonas aeruginosaRESUMO
Invasion and metastasis are the leading causes of death in patients with breast cancer (BC), and epithelial-mesenchymal transformation (EMT) plays an essential role in this process. Here, we found that Lnc-408, a novel long noncoding RNA (lncRNA), is significantly upregulated in BC cells undergoing EMT and in BC tumor with lymphatic metastases compared with those without lymphatic metastases. Lnc-408 can enhance BC invasion and metastasis by regulating the expression of LIMK1. Mechanistically, Lnc-408 serves as a sponge for miR-654-5p to relieve the suppression of miR-654-5p on its target LIMK1. Knockdown or knockout of Lnc-408 in invasive BC cells clearly decreased LIMK1 levels, and ectopic Lnc-408 in MCF-7 cells increased LIMK1 expression to promote cell invasion. Lnc-408-mediated enhancement of LIMK1 plays a key role in cytoskeletal stability and promotes invadopodium formation in BC cells via p-cofilin/F-actin. In addition, the increased LIMK1 also facilitates the expression of MMP2, ITGB1, and COL1A1 by phosphorylating CREB. In conclusion, our findings reveal that Lnc-408 promotes BC invasion and metastasis via the Lnc-408/miR-654-5p/LIMK1 axis, highlighting a novel promising target for the diagnosis and treatment of BC.
Assuntos
Neoplasias da Mama/genética , Movimento Celular/genética , Quinases Lim/genética , Metástase Neoplásica/genética , RNA Longo não Codificante/genética , Apoptose/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células/genética , Transição Epitelial-Mesenquimal/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Células MCF-7 , MicroRNAs/genética , Metástase Neoplásica/patologia , Regulação para Cima/genéticaRESUMO
Tumor initiation, development, and relapse may be closely associated with cancer stem cells (CSCs). The complicated mechanisms underlying the maintenance of CSCs are keeping in illustration. Long noncoding RNAs (lncRNAs), due to their multifunction in various biological processes, have been indicated to play a crucial role in CSC renewal and stemness maintenance. Using lncRNA array, we identified a novel lncRNA (named lnc408) in epithelial-mesenchymal transition-related breast CSCs (BCSCs). The lnc408 is high expressed in BCSCs in vitro and in vivo. The enhanced lnc408 is critical to BCSC characteristics and tumorigenesis. Lnc408 can recruit transcript factor SP3 to CBY1 promoter to serve as an inhibitor in CBY1 transcription in BCSCs. The high expressed CBY1 in non-BCSC interacts with 14-3-3 and ß-catenin to form a ternary complex, which leads a translocation of the ternary complex into cytoplasm from nucleus and degradation of ß-catenin in phosphorylation-dependent pattern. The lnc408-mediated decrease of CBY1 in BCSCs impairs the formation of 14-3-3/ß-catenin/CBY1 complex, and keeps ß-catenin in nucleus to promote CSC-associated CD44, SOX2, Nanog, Klf4, and c-Myc expressions and contributes to mammosphere formation; however, restoration of CBY1 expression in tumor cells reduces BCSC and its enrichment, thus lnc408 plays an essential role in maintenance of BCSC stemness. In shortly, these findings highlight that the novel lnc408 functions as an oncogenic factor by recruiting SP3 to inhibit CBY1 expression and ß-catenin accumulation in nucleus to maintain stemness properties of BCSCs. Lnc408-CBY1-ß-catenin signaling axis might serve as a new diagnostic and therapeutic target for breast cancer.
Assuntos
Neoplasias da Mama/metabolismo , Proteínas de Transporte/metabolismo , Células-Tronco Neoplásicas/metabolismo , Proteínas Nucleares/metabolismo , Fator de Transcrição Sp3/metabolismo , beta Catenina/metabolismo , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Feminino , Xenoenxertos , Humanos , Fator 4 Semelhante a Kruppel , Camundongos , Camundongos Nus , Células-Tronco Neoplásicas/patologiaRESUMO
BACKGROUND: This study examined (a) the relationship that interpersonal problems and depression share with the intensity of mobile phone use and (b) their effects on suicide-related behaviors (SRBs) and self-harming behaviors among adolescents. METHODS: In this cross-sectional study, adolescents were recruited from 11 middle and high schools in Shenzhen, China. They completed self-reported measures that assessed mobile phone use, their history of SRBs (i.e., suicidal ideation, suicidal planning, and attempted suicide) and self-harming behaviors, depression, and interpersonal problems. RESULTS: Interpersonal problems and depression mediated the relationship between high-intensity mobile phone use and the risk for SRBs and self-harming behaviors among Chinese adolescents. CONCLUSIONS: The findings delineate the pathways through which interpersonal problems and depression increase the risk for SRBs and self-harming behaviors among adolescents who report high-intensity mobile phone use. Future studies should develop interventions that alleviate interpersonal problems and depressive symptoms to reduce the risk for SRBs and self-harming behaviors among adolescents who report high-intensity mobile phone use.
Assuntos
Uso do Telefone Celular , Ideação Suicida , Adolescente , China/epidemiologia , Estudos Transversais , Depressão/epidemiologia , Humanos , Fatores de RiscoRESUMO
Metastasis is the leading cause of breast cancer-related deaths. Cancer-associated fibroblasts (CAFs), the predominant stromal cell type in the breast tumour microenvironment, may contribute to cancer progression through interaction with tumour cells. Nonetheless, little is known about the details of the underlying mechanism. Here we found that interaction of interleukin 32 (IL32) with integrin ß3 (encoded by ITGB3; a member of the integrin family) mediating the cross-talk between CAFs and breast cancer cells plays a crucial role in CAF-induced breast tumour invasiveness. IL32, an 'RGD' motif-containing cytokine, was found to be abundantly expressed in CAFs. Integrin ß3 turned out to be up-regulated in breast cancer cells during epithelial-mesenchymal transition (EMT). CAF-derived IL32 specifically bound to integrin ß3 through the RGD motif, thus activating intracellular downstream p38 MAPK signalling in breast cancer cells. This signalling increased the expression of EMT markers (fibronectin, N-cadherin, and vimentin) and promoted tumour cell invasion. Counteracting IL32 activity, a knockdown of IL32 or integrin ß3 led to specific inactivation of p38 MAPK signalling in tumour cells. Blockage of the p38 MAPK pathway also diminished IL32-induced expression of EMT markers and breast cancer cell invasion and metastasis. Thus, our data indicate that CAF-secreted IL32 promotes breast cancer cell invasion and metastasis via integrin ß3-p38 MAPK signalling.
Assuntos
Neoplasias da Mama/enzimologia , Fibroblastos Associados a Câncer/metabolismo , Movimento Celular , Integrina beta3/metabolismo , Interleucinas/metabolismo , Comunicação Parácrina , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Antígenos CD/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Caderinas/metabolismo , Fibroblastos Associados a Câncer/patologia , Técnicas de Cocultura , Transição Epitelial-Mesenquimal , Feminino , Fibronectinas/metabolismo , Humanos , Integrina beta3/genética , Interleucinas/genética , Células MCF-7 , Camundongos Nus , Invasividade Neoplásica , Metástase Neoplásica , Oligopeptídeos/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Transdução de Sinais , Vimentina/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Tamoxifen resistance is a major clinical challenge in breast cancer treatment. Our previous studies find that GPER and its down-stream signaling play a pivotal role in the development of tamoxifen (TAM) resistance. cDNA array analysis indicated a set of genes associated with cell apoptosis are aberrant in GPER activated and TAM-resistant MCF-7R cells compared with TAM-sensitive MCF-7 cells. Among these genes, Bim (also named BCL2-L11), a member of the BH3-only pro-apoptotic protein family is significantly decreased, and TRIM RING ï¬nger protein TRIM2 (a ubiquitin ligase) is highly expressed in MCF-7R. To understand the mechanism of TAM-resistance in GPER activated ER+ breast cancer, the function of TRIM2 and Bim inducing cell apoptosis was studied. By using immunohistochemical and western blot analysis, there is an adverse correlation between TRIM2 and Bim in TAM-resistant breast tumor tissues and MCF-7R cells. Knockdown Bim in TAM-sensitive MCF-7 cells or overexpression of Bim in TAM-resistant MCF-7 cells significantly changed its sensibility to TAM through altering the levels of cleaved PARP and caspase-3. Activation of GPER and its downstream signaling MAPK/ERK, not PI3K/AKT, led to enhanced TRIM2 protein levels and affected the binding between TRIM2 and Bim which resulted in a reduced Bim in TAM-resistant breast cancer cells. Thus, the present study provides a novel insight to TAM-resistance in ER-positive breast cancer cells.
Assuntos
Proteína 11 Semelhante a Bcl-2/genética , Neoplasias da Mama/metabolismo , Resistência a Medicamentos , Proteínas Nucleares/genética , Receptores de Estrogênio/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Apoptose , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Sistema de Sinalização das MAP Quinases , Células MCF-7 , Proteínas Nucleares/metabolismo , Receptores de Estrogênio/genética , Receptores Acoplados a Proteínas G/genética , Tamoxifeno/farmacologiaRESUMO
Drosha is an RNA III-like enzyme that has an aberrant expression in some tumors. Our previous studies showed the aberrant Drosha in gastric tumors. However, the roles of nuclear Drosha, the main regulator of microRNA (miRNA) biogenesis, in gastric cancer (GC) progression remain poorly understood. In this study, we demonstrated that nuclear Drosha is significantly associated with cell invasion of GC and that Drosha silence impedes the tumor invasion. Knockdown of Drosha led to a set of dysregulated miRNAs in GC cells. Multiple targets of these miRNAs were the members in cell migration, invasion and metastasis-associated signaling (e.g. ECM-receptor interaction, focal adhesion, p53 signaling and MAPK signaling pathway) revealed by bioinformatics analysis. LAMC2 (a key element of ECM-receptor signaling) and CD82 (a suppressor of p53 signaling) are the targets of miR-622 and miR-197, respectively. High levels of LAMC2 and low levels of CD82 were significantly related to the worse outcome for GC patients. Furthermore, overexpression of LAMC2 and knockdown of CD82 markedly promoted GC cell invasion and activated EGFR/ERK1/2-MMP7 signaling via upregulation of the expression of phosphorylated (p)-EGFR, p-ERK1/2 and MMP7. Our findings suggest that nuclear Drosha potentially has a role in the development of GC.
Assuntos
Receptores ErbB/metabolismo , Proteína Kangai-1/metabolismo , Laminina/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Metaloproteinase 7 da Matriz/metabolismo , MicroRNAs/metabolismo , Ribonuclease III/metabolismo , Neoplasias Gástricas/metabolismo , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Invasividade Neoplásica/patologia , Metástase Neoplásica/patologia , Fosforilação/fisiologia , Transdução de Sinais/fisiologia , Neoplasias Gástricas/patologia , Regulação para Cima/fisiologiaRESUMO
Objective To establish a gastric cancer cell line with stable Drosha silenced and explore the effect of Drosha on the chemosensitivity of gastric cancer cells to epirubicin. Methods Interfering sequences targeting Drosha were designed and inserted into the lentiviral vectors, which were used to transfect MGC-803 cells. The level of Drosha mRNA was detected by quantitative real-time PCR; Drosha protein was detected by Western blotting; MTT assay was performed to test the 50% inhibitory concentration (IC50) of epirubicin agaisnt wide-type MGC-803 cells. After the treatment with IC50 epirubicin, the apoptosis rate of each cell group was determined by flow cytometry; the expressions of apoptosis-related proteins caspase-3, caspase-9, Bax, Bcl-2 were assessed by Western blotting. Results The gastric cancer MGC-803 cells with stable Drosha silenced were successfully established, and the levels of Drosha mRNA and protein were reduced. After the cells were treated with 0.5 mg/L(IC50) epirubicin, the apoptosis rate of MGC-803 cells was raised, the protein expressions of caspase-3 , caspase-9 and Bax were significantly upregulated and Bcl-2 was downregulated. Conclusion The silence of Drosha expression can promote the sensitivity of gastric cancer to epirubicin.
Assuntos
Antineoplásicos/farmacologia , Epirubicina/farmacologia , Ribonuclease III/genética , Neoplasias Gástricas/genética , Apoptose/efeitos dos fármacos , Caspase 9/genética , Caspase 9/metabolismo , Linhagem Celular Tumoral , Regulação para Baixo/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ribonuclease III/metabolismo , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/fisiopatologia , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismoRESUMO
Cancer stem cells (CSCs) are a subpopulation of neoplastic cells with self-renewal capacity and limitless proliferative potential as well as high invasion and migration capacity. These cells are commonly associated with epithelial-mesenchymal transition (EMT), which is also critical for tumor metastasis. Recent studies illustrate a direct link between EMT and stemness of cancer cells. Long non-coding RNAs (lncRNAs) have emerged as important new players in the regulation of multiple cellular processes in various diseases. To date, the role of lncRNAs in EMT-associated CSC stemness acquisition and maintenance remains unclear. In this study, we discovered that a set of lncRNAs were dysregulated in Twist-positive mammosphere cells using lncRNA microarray analysis. Multiple lncRNAs-associated canonical signaling pathways were identified via bioinformatics analysis. Especially, the Shh-GLI1 pathway associated lncRNA-Hh, transcriptionally regulated by Twist, directly targets GAS1 to stimulate the activation of hedgehog signaling (Hh). The activated Hh increases GLI1 expression, and enhances the expression of SOX2 and OCT4 to play a regulatory role in CSC maintenance. Thus, the mammosphere-formation efficiency (MFE) and the self-renewal capacity in vitro, and oncogenicity in vivo in Twist-positive breast cancer cells are elevated. lncRNA-Hh silence in Twist-positive breast cells attenuates the activated Shh-GLI1 signaling and decreases the CSC-associated SOX and OCT4 levels, thus reduces the MFE and tumorigenesis of transplanted tumor. Our results reveal that lncRNAs function as an important regulator endowing Twist-induced EMT cells to gain the CSC-like stemness properties.
Assuntos
Neoplasias da Mama/metabolismo , Proteínas Hedgehog/metabolismo , Células-Tronco Neoplásicas/metabolismo , Proteínas Nucleares/metabolismo , RNA Longo não Codificante/metabolismo , Transdução de Sinais , Proteína 1 Relacionada a Twist/metabolismo , Animais , Neoplasias da Mama/patologia , Carcinogênese/patologia , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal/genética , Feminino , Humanos , Camundongos Endogâmicos BALB C , Camundongos Nus , Células-Tronco Neoplásicas/patologia , RNA Longo não Codificante/genética , Esferoides Celulares/patologiaRESUMO
Twist, a highly conserved basic Helix-Loop-Helix transcription factor, functions as a major regulator of epithelial-mesenchymal transition (EMT) and tumor metastasis. In different cell models, signaling pathways such as TGF-ß, MAPK/ERK, WNT, AKT, JAK/STAT, Notch, and P53 have also been shown to play key roles in the EMT process, yet little is known about the signaling pathways regulated by Twist in tumor cells. Using iTRAQ-labeling combined with 2D LC-MS/MS analysis, we identified 194 proteins with significant changes of expression in MCF10A-Twist cells. These proteins reportedly play roles in EMT, cell junction organization, cell adhesion, and cell migration and invasion. ECM-receptor interaction, MAPK, PI3K/AKT, P53 and WNT signaling were found to be aberrantly activated in MCF10A-Twist cells. Ingenuity Pathways Analysis showed that integrin ß1 (ITGB1) acts as a core regulator in linking integrin-linked kinase (ILK), Focal-adhesion kinase (FAK), MAPK/ERK, PI3K/AKT, and WNT signaling. Increased Twist and ITGB1 are associated with breast tumor progression. Twist transcriptionally regulates ITGB1 expression. Over-expression of ITGB1 or Twist in MCF10A led to EMT, activation of FAK/ILK, MAPK/ERK, PI3K/AKT, and WNT signaling. Knockdown of Twist or ITGB1 in BT549 and Hs578T cells decreased activity of FAK, ILK, and their downstream signaling, thus specifically impeding EMT and cell invasion. Knocking down ILK or inhibiting FAK, MAPK/ERK, or PI3K/AKT signaling also suppressed Twist-driven EMT and cell invasion. Thus, the Twist-ITGB1-FAK/ILK pathway and their downstream signaling network dictate the Twist-induced EMT process in human mammary epithelial cells and breast cancer cells.
Assuntos
Movimento Celular , Transição Epitelial-Mesenquimal , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Integrina beta1/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Proteína 1 Relacionada a Twist/metabolismo , Linhagem Celular Tumoral , Humanos , Glândulas Mamárias Humanas/citologia , Invasividade Neoplásica , PrognósticoRESUMO
BACKGROUND: The nuclear localization of Drosha is critical for its function as a microRNA maturation regulator. Dephosphorylation of Drosha at serine 300 and serine 302 disrupts its nuclear localization, and aberrant distribution of Drosha has been detected in some tumors. AIMS: The purpose of the present study was to assess cytoplasmic/nuclear Drosha expression in gastric cancer carcinogenesis and progression. METHODS: Drosha expression and its subcellular location was investigated by immunohistochemical staining of a set of tissue microarrays composed of normal adjacent tissues (374), chronic gastritis (137), precancerous lesions (94), and gastric adenocarcinoma (829) samples, and in gastric cancer cell lines with varying differentiation by immunofluorescence and western blot assay. RESULTS: Gradual loss of cytoplasmic Drosha was accompanied by tumor progression in both gastric cancer tissues and cell lines, and was inversely associated with tumor volume (P = 0.002), tumor grade (P < 0.001), tumor stage (P = 0.018), and distant metastasis (P = 0.026). Aberrant high levels of cytoplasmic Drosha were apparent in intestinal metaplasia and dysplasia tissues. The levels of nuclear Drosha were sharply decreased in chronic gastritis and maintained through precancerous lesions to gastric cancer. High levels of cytoplasmic Drosha predicted longer survival (LR = 7.088, P = 0.008) in gastric cancer patients. CONCLUSIONS: Our data provide novel insights into gastric cancer that cytoplasmic Drosha potentially plays a role in preventing carcinogenesis and tumor progression, and may be an independent predictor of patient outcome.
Assuntos
Carcinoma/metabolismo , Gastrite Atrófica/metabolismo , Lesões Pré-Cancerosas/metabolismo , Ribonuclease III/metabolismo , Neoplasias Gástricas/metabolismo , Carcinoma/mortalidade , Carcinoma/patologia , Estudos de Casos e Controles , Linhagem Celular Tumoral , China/epidemiologia , Citoplasma/metabolismo , Progressão da Doença , Feminino , Mucosa Gástrica/metabolismo , Humanos , Masculino , Metaplasia/metabolismo , Pessoa de Meia-Idade , Prognóstico , Estômago/patologia , Neoplasias Gástricas/mortalidade , Neoplasias Gástricas/patologia , Análise Serial de TecidosRESUMO
OBJECTIVE: To investigate the role of Drosha expression in the progression of gastric adenocarcinoma and its impact on the invasive ability of SGC-7901 human gastric cancer cells. METHODS: Drosha expression was detected in 889 gastric carcinoma samples on tissue microarrays by immunohistochemistry staining and quantified by Image-Pro Plus software. Statistical analysis was used to evaluate the correlations between Drosha expression and the clinicopathological characteristics of the 889 tumor cases or the outcomes of 309 gastric adenocarcinoma patients. Drosha was knocked down in SGC-7901 cells by small interfering RNA (siRNA), and cell invasive ability was assessed by Transwell(TM) assay. RESULTS: Drosha expression was the highest in the well differentiated gastric adenocarcinoma (median absorbance, 0.4195), and the lowest in the poorly differentiated samples. Drosha expression was significantly related to Laren classification, tumor size, tumor invasion depth, lymph node metastasis, tumor pathological grade and stage. Patients in Drosha positive group had a higher survival rate than those in Drosha negative group. Silencing Drosha in SGC-7901 cells resulted in an enhanced cell invasion. CONCLUSION: Drosha expression was reduced gradually with the degrading histological differentiation of gastric adenocarcinoma, and the knock-down of Drosha expression could promote gastric adenocarcinoma cell invasion.
Assuntos
Adenocarcinoma/patologia , Ribonuclease III/fisiologia , Neoplasias Gástricas/patologia , Adenocarcinoma/química , Adulto , Idoso , Humanos , Pessoa de Meia-Idade , Invasividade Neoplásica , Ribonuclease III/análise , Neoplasias Gástricas/químicaRESUMO
Twist, a key regulator of epithelial-mesenchymal transition (EMT), plays an important role in the development of a tumorigenic phenotype. Energy metabolism reprogramming (EMR), a newly discovered hallmark of cancer cells, potentiates cancer cell proliferation, survival, and invasion. Currently little is known about the effects of Twist on tumor EMR. In this study, we found that glucose consumption and lactate production were increased and mitochondrial mass was decreased in Twist-overexpressing MCF10A mammary epithelial cells compared with vector-expressing MCF10A cells. Moreover, these Twist-induced phenotypic changes were augmented by hypoxia. The expression of some glucose metabolism-related genes such as PKM2, LDHA, and G6PD was also found to be upregulated. Mechanistically, activated ß1-integrin/FAK/PI3K/AKT/mTOR and suppressed P53 signaling were responsible for the observed EMR. Knockdown of Twist reversed the effects of Twist on EMR in Twist-overexpressing MCF10A cells and Twist-positive breast cancer cells. Furthermore, blockage of the ß1-integrin/FAK/PI3K/AKT/mTOR pathway by siRNA or specific chemical inhibitors, or rescue of p53 activation can partially reverse the switch of glucose metabolism and inhibit the migration of Twist-overexpressing MCF10A cells and Twist-positive breast cancer cells. Thus, our data suggest that Twist promotes reprogramming of glucose metabolism in MCF10A-Twist cells and Twist-positive breast cancer cells via activation of the ß1-integrin/FAK/PI3K/AKT/mTOR pathway and inhibition of the p53 pathway. Our study provides new insight into EMR.
Assuntos
Neoplasias da Mama/enzimologia , Metabolismo Energético , Glucose/metabolismo , Proteínas Nucleares/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Proteína Supressora de Tumor p53/metabolismo , Proteína 1 Relacionada a Twist/metabolismo , Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Movimento Celular , Metabolismo Energético/efeitos dos fármacos , Metabolismo Energético/genética , Transição Epitelial-Mesenquimal , Feminino , Quinase 1 de Adesão Focal/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Integrina beta1/metabolismo , Ácido Láctico/metabolismo , Mitocôndrias/enzimologia , Mitocôndrias/patologia , Proteínas Nucleares/genética , Fenótipo , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Interferência de RNA , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Fatores de Tempo , Transfecção , Proteína Supressora de Tumor p53/genética , Proteína 1 Relacionada a Twist/genéticaRESUMO
Abnormal proliferation is one characteristic of cancer-associated fibroblasts (CAFs), which play a key role in tumorigenesis and tumor progression. Oxidative stress (OS) is the root cause of CAFs abnormal proliferation. ATM (ataxia-telangiectasia mutated protein kinase), an important redox sensor, is involved in DNA damage response and cellular homeostasis. Whether and how oxidized ATM regulating CAFs proliferation remains unclear. In this study, we show that there is a high level of oxidized ATM in breast CAFs in the absence of double-strand breaks (DSBs) and that oxidized ATM plays a critical role in CAFs proliferation. The effect of oxidized ATM on CAFs proliferation is mediated by its regulation of cellular redox balance and the activity of the ERK, PI3K-AKT, and Wnt signaling pathways. Treating cells with antioxidant N-acetyl-cysteine (NAC) partially rescues the proliferation defect of the breast CAFs caused by ATM deficiency. Administrating cells with individual or a combination of specific inhibitors of the ERK, PI3K-AKT, and Wnt signaling pathways mimics the effect of ATM deficiency on breast CAF proliferation. This is mainly ascribed to the ß-catenin suppression and down-regulation of c-Myc, thus further leading to the decreased cyclinD1, cyclinE, and E2F1 expression and the enhanced p21(Cip1) level. Our results reveal an important role of oxidized ATM in the regulation of the abnormal proliferation of breast CAFs. Oxidized ATM could serve as a potential target for treating breast cancer.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Neoplasias da Mama/metabolismo , Fibroblastos/metabolismo , Via de Sinalização Wnt , Antioxidantes/química , Linhagem Celular Tumoral , Proliferação de Células , Quebras de DNA de Cadeia Dupla , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Homeostase , Humanos , MAP Quinase Quinase 1/metabolismo , Mitocôndrias/metabolismo , Oxirredução , Estresse Oxidativo , Oxigênio/química , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Interferente Pequeno/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , beta Catenina/metabolismoRESUMO
OBJECTIVE: To investigate the difference of miRNA expression levels of cancer-associated fibroblasts (CAFs) and normal fibroblasts (NFs) in human breast cancer microenvironment and its effect on the biological features of CAFs. METHODS: Collagenase-1 was used to digest the cancer and adjacent tissues to isolate CAFs and NFs. The isolated cells were cultured and characterized in purity and biological features. The expression of fibroblast secretory protein (FSP) in CAFs and NFs was detected by immunofluorescence staining and Western blotting. Transwell(TM) assay was adopted to compare the invasion ability of CAFs and NFs. The different expressions of miRNAs in CAFs versus NFs were detected by miRNA microarray and analyzed by Significance Analysis of Microarrays (SAM). The differences in miR-205 and miR-221 expressions were verified by real-time quantitative PCR (qRT-PCR). The common target genes of the miRNAs were predicted using multi-bioinformatics tools. The pathway analysis was conducted through the Database for Annotation, Visualization and Integrated Discovery (DAVID) v6.7. The secreting products of TGF-ß or IL-6 signaling pathway, matrix metalloproteinase (MMP)-1, MMP-2 and MMP-9 were analyzed by ELISA. RESULTS: The primary CAFs and NFs were isolated from breast cancer patients with a purity of over 95%. Compared with NFs, the expression of FSP was obviously elevated in CAFs, and the invasion ability of CAFs was enhanced. The miRNA microarray results showed that there were 10 miRNA genes dysregulated in CAFs, including 3 up-regulated (miR-221-5p, miR-31-3p, miR-221-3p) and 7 down-regulated genes (miR-205, miR-200b , miR-200c, miR-141, miR-101, miR-342-3p, let-7g). The common targets genes of the dysregulated miRNAs were mainly focused on HGF, chemokine signaling, insulin signaling, MAPK signaling, tight junction signaling, adherence junction signaling, EGF1 signaling, androgen-receptor signaling, Wnt and IL-7 signaling. In addition, dysregulated miR-200b/c and miR-141 et al. affect TGF-ß and IL-6 signaling through inhibiting their target genes in CAFs, thus promoting invasion and migration of CAFs. CONCLUSION: The miRNA expression profile was markedly dysregulated in CAFs. Those dysregulated miRNAs may take part in the transformation from NFs to CAFs, and also have a close relationship with adhesion, migration, proliferation, secretion and cell-cell interaction of CAFs.