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We aimed to develop and validate a novel combined score to improve the assessment of liver fibrosis progression in patients with chronic hepatitis B (CHB). In this study, a total of 331 CHB patients from three cohorts who underwent liver biopsy were enrolled, and the Scheuer system was used for liver fibrosis classification. The combined score was derived by principal component analysis of key differentially expressed genes. For significant liver fibrosis (≥S2), the areas under the receiver operating characteristics curves (AUROCs) of the combined score were 0.838, 0.842, and 0.881 in the three cohorts, respectively. And for advanced liver fibrosis (≥S3), the AUROCs were 0.794, 0.801, and 0.901, respectively. Compared with the results of AUROCs for aspartate aminotransferase≥to≥platelet ratio (APRI) and fibrosis index based on four factors (FIB-4) in the validation cohorts, better clinical diagnostic value for assessing the progression of liver fibrosis was found in the combined score. Additionally, univariate ordered logistic regression analysis indicated that the combined score could serve as a more superior and stable risk factor than APRI and FIB-4 in the assessment of liver fibrosis. For CHB patients with normal alanine aminotransferase (ALT), our results further emphasized the diagnostic value of the combined score for significant fibrosis (≥S2) and advanced fibrosis (≥S3). Moreover, it was found that patients with the high combined score, who were associated with the advanced fibrosis stage, had higher levels of drug sensitivity and immune checkpoint expression. In conclusion, the novel combined score could serve as a potential biomarker and contribute to improving the assessment of fibrosis stage in CHB patients.
Assuntos
Hepatite B Crônica , Humanos , Aspartato Aminotransferases , Biomarcadores , Biópsia , Plaquetas/patologia , Hepatite B Crônica/complicações , Hepatite B Crônica/diagnóstico , Hepatite B Crônica/patologia , Cirrose Hepática/patologia , Contagem de Plaquetas , Estudos Retrospectivos , Curva ROC , Índice de Gravidade de DoençaRESUMO
It is known that ribonucleotide reductase M2 (RRM2) could be induced by hepatitis B virus (HBV) via DNA damage response. However, whether RRM2 is a potential biomarker for diagnosing and monitoring liver fibrosis in chronic hepatitis B (CHB) patients is still unclear. In this study, CHB patients from GSE84044 (a transcriptome data from GEO data set) were downloaded and RRM2 was selected as a hub gene. Interestingly, a positive correlation was found between serum RRM2 and liver fibrosis stage. The similar results were found in CHB patients with normal alanine aminotransferase (ALT). Notably, RRM2 could effectively differentiate preliminary fibrosis from advanced fibrosis in CHB patients with/without normal ALT. In addition, RRM2 had a better performance in diagnosing liver fibrosis than two commonly used noninvasive methods (aspartate aminotransferase-to-platelet ratio index and fibrosis index based on the four factors), two classic fibrotic biomarkers (hyaluronic acid and type IV collagen) as well as Mac-2 binding protein glycosylation isomer, a known serum fibrosis marker. Moreover, CHB patients with high RRM2, who were associated with advanced fibrosis, had higher expressions of immune checkpoints. Overall, serum RRM2 may be a promising biomarker for diagnosing and monitoring liver fibrosis in CHB patients.
Assuntos
Hepatite B Crônica , Humanos , Hepatite B Crônica/complicações , Hepatite B Crônica/diagnóstico , Curva ROC , Cirrose Hepática , Fígado/patologia , Vírus da Hepatite B , Fibrose , Biomarcadores , Alanina TransaminaseRESUMO
As a highly heterogeneous cancer, the prognostic stratification and personalized management of hepatocellular carcinoma (HCC) are still challenging. Recently, Antigen-presenting-cells (APCs) and T-cells-infiltration (TCI) have been reported to be implicated in modifying immunology in HCC. Nevertheless, the clinical value of APCs and TCI-related long non-coding RNAs (LncRNAs) in the clinical outcomes and precision treatment of HCC is still obscure. In this study, a total of 805 HCC patients were enrolled from three public datasets and an external clinical cohort. 5 machine learning (ML) algorithms were transformed into 15 kinds of ML integrations, which was used to construct the preliminary APC-TCI related LncRNA signature (ATLS). According to the criterion with the largest average C-index in the validation sets, the optimal ML integration was selected to construct the optimal ATLS. By incorporating several vital clinical characteristics and molecular features for comparison, ATLS was demonstrated to have a relatively more significantly superior predictive capacity. Additionally, it was found that the patients with high ATLS score had dismal prognosis, relatively high frequency of tumor mutation, remarkable immune activation, high expression levels of T cell proliferation regulators and anti-PD-L1 response as well as extraordinary sensitivity to Oxaliplatin/Fluorouracil/Lenvatinib. In conclusion, ATLS may serve as a robust and powerful biomarker for improving the clinical outcomes and precision treatment of HCC.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , RNA Longo não Codificante , Humanos , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/terapia , RNA Longo não Codificante/genética , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/terapia , Linfócitos T , Aprendizado de Máquina , PrognósticoRESUMO
Necroptosis has been reported to be involved in cancer progression and associated with cancer prognosis. However, the prognostic values of necroptosis-related genes (NRGs) in hepatocellular carcinoma (HCC) remain largely unknown. This study aimed to build a signature on the basis of NRGs to evaluate the prognosis of HCC patients. In this study, using bioinformatic analyses of transcriptome sequencing data of HCC (n = 370) from The Cancer Genome Atlas (TCGA) database, 63 differentially expressed NRGs between HCC and adjacent normal tissues were determined. 24 differentially expressed NRGs were found to be related with overall survival (OS). Seven optimum NRGs, determined using Lasso regression and multivariate Cox regression analysis, were used to construct a new prognostic risk signature for predicting the prognosis of HCC patients. Then survival status scatter plots and survival curves demonstrated that the prognosis of patients with high-Riskscore was worse. The prognostic value of this 7-NRG signature was validated by the International Cancer Genome Consortium (ICGC) cohort and a local cohort (Wenzhou, China). Notably, Riskscore was defined as an independent risk factor for HCC prognosis using multivariate cox regression analysis. Immune infiltration analysis suggested that higher macrophage infiltration was found in patients in the high-risk group. Finally, enhanced 7 NRGs were found in HCC tissues by immunohistochemistry. In conclusion, a novel 7-NRG prognostic risk signature is generated, which contributes to the prediction in the prognosis of HCC patients for the clinicians.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/genética , Necroptose/genética , Neoplasias Hepáticas/genética , China , Biologia Computacional , PrognósticoRESUMO
BACKGROUND: Exosomes play an important role in the tumor microenvironment (TME) and the mechanisms of tumor immune escape in hepatocellular carcinoma (HCC). It is known that immunosuppressive genes, involved in the processes of tumor immunosuppression, are associated with cancer progression. This study aimed to explore the prognostic values of exosome-related immunosuppression genes (ERIGs) in HCC. METHODS: The RNA-seq transcriptome data of 374 HCC patients were obtained from the Cancer Genome Atlas (TCGA) database. The TCGA cohort was randomly divided into the training cohort and validation cohort in a 1:1 ratio. WGCNA analysis and Pearson correlation analysis were used to identify ERIGs. The Lasso regression method was used to construct a 5-ERIG signature. The prognostic value of our signature was examined in the First Affiliated Hospital of Wenzhou Medical University (FAHWMU) cohort. RESULTS: Univariate Cox regression analysis was used to screen prognostic ERIGs. Subsequently, these prognostic ERIGs were included in Lasso regression analyses to identify 5 key ERIGs (ASAP1, IARS1, GTF3C2, TPD5L2 and SLC52A2) and construct a 5-ERIG signature. The patients in the low-risk group had better prognosis than those in the high-risk group. Univariate and multivariate cox regression revealed that risk score was an independent prognostic risk factor of HCC. Gene set enrichment analysis (GSEA) showed that this signature was highly associated with TME-related pathways. Subsequent analyses revealed the potential role of the signature in regulating the TME in HCC. In addition, a lower immunotherapy score was found in patients with high risk-score. Of note, this signature was confirmed to have a good performance in predicting HCC prognosis in the FAHWMU cohort. Moreover, knockdown of 5 ERIGs of this signature contributed to the suppression the Hep3B cell proliferation. CONCLUSIONS: We generated a novel prognostic 5-ERIG signature to accurately predict the prognosis of patients with HCC, and this signature may serve as an indicator of immunotherapy for HCC.
Assuntos
Carcinoma Hepatocelular , Exossomos , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/genética , Exossomos/genética , Microambiente Tumoral/genética , Neoplasias Hepáticas/genética , Terapia de Imunossupressão , Hospitais Universitários , PrognósticoRESUMO
LncRNA N6-methylandenosine (m6A) modification has been shown to be associated with the constitution of the tumor microenvironment (TME) and tumorigenesis. It's essential to understand the mechanisms of lncRNA m6A modification in hepatocellular carcinoma (HCC) and identify relative prognostic predictors to guide therapy and explore potential therapeutic targets. Pearson correlation analysis was performed to identify m6A-related lncRNAs in 374 patients with HCC. Unsupervised cluster analysis of the potential m6A-related lncRNA-based HCC subtypes was conducted, followed by the concurrent analysis of their relationship with TME characteristics, immune checkpoints, immune features, and prognosis through single sample gene set enrichment analysis and ESTIMATE algorithm. Cox regression analyses were performed to screen prognostic m6A-related lncRNA, construct an m6A-related lncRNA signature (m6A-RLRS), and establish an integrated nomogram for the prognosis of patients with HCC. We identified 61 m6A-related lncRNAs and two HCC subtypes defined by consensus cluster of m6A-related lncRNAs with distinct clinical features. Progression-free survival (PFS), three TME-related scores, 15 immune-associated gene sets, and two immune checkpoints expression were found to be significantly different among the two subtypes. Twenty-five prognostic m6A-related lncRNAs were determined, four of which were included to establish an m6A-RLRS with favorable discrimination, and the signature was validated in the validation set and an independent FAHWMU cohort (n = 60). Furthermore, a novel nomogram combining signature and clinical predictors was generated with a C-index of 0.703, and an original ceRNA regulatory network consisting of 9 lncRNAs, 28 miRNAs, and 75 target mRNAs was constructed. Finally, the differential expression of four m6A-related lncRNA was verified by qRT-PCR. In conclusion, m6A-related lncRNA prognostic signature and molecular subtype contributes to accurately predict the prognosis of HCC and provide potential novel therapeutic targets.
Assuntos
Carcinoma Hepatocelular , Leucemia Eritroblástica Aguda , Neoplasias Hepáticas , RNA Longo não Codificante , Biomarcadores Tumorais/metabolismo , Carcinoma Hepatocelular/patologia , Regulação Neoplásica da Expressão Gênica , Humanos , Estimativa de Kaplan-Meier , Neoplasias Hepáticas/patologia , Prognóstico , RNA Longo não Codificante/metabolismo , Microambiente Tumoral/genéticaRESUMO
Liver fibrosis, which results from chronic liver injury due to factors such as chronic alcohol consumption, hepatitis virus infections, and immune attacks, is marked by excessive deposition of extracellular matrix (ECM). Resveratrol (Res), a polyphenol phytoalexin, has been demonstrated to show anti-inflammatory, antioxidative, antiproliferative, and chemopreventive activities. In recent years, Res has been found to inhibit liver fibrosis. Enhanced Hippo pathway activation has also been reported to inhibit tumor progression and liver fibrosis. In the present study, the role of the Hippo pathway in mediating the effects of Res on hepatic stellate cells (HSCs) was examined. We found that Res significantly suppresses HSC proliferation, reducing the cell index. Res induced HSC inactivation, reducing collagen deposition and α-smooth muscle actin (α-SMA) expression. In addition, Res contributed to HSC apoptosis, upregulating Bax and downregulating Bcl-2 expression. Notably, the Hippo pathway was involved in the Res-mediated suppression of HSC activation. Res enhanced the activation of the Hippo pathway and reduced yes-associated protein (YAP) and transcriptional coactivator with the PDZ-binding motif (TAZ) expression. Interestingly, the YAP overexpression inhibited Res-induced HSC inactivation and apoptosis. In conclusion, these results demonstrate that Res inhibits HSC activation, at least in part, via the Hippo pathway. The present study indicates a new antifibrotic mechanism of Res and provides novel insights into Hippo-mediated HSC apoptosis and HSC activation in liver fibrosis.
Assuntos
Células Estreladas do Fígado/efeitos dos fármacos , Via de Sinalização Hippo/efeitos dos fármacos , Resveratrol/farmacologia , Animais , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Transdiferenciação Celular/efeitos dos fármacos , Células Cultivadas , Colágeno/metabolismo , Células Estreladas do Fígado/fisiologia , Via de Sinalização Hippo/fisiologia , Humanos , Cirrose Hepática/tratamento farmacológico , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Resveratrol/uso terapêutico , Proteínas de Sinalização YAP/fisiologiaRESUMO
Thymoquinone (TQ), the main active constituent of Nigella sativa seeds, has been shown to play a role in antioxidation, anti-inflammation, and antitumor. Recent studies have demonstrated that TQ contributes to the suppression of liver fibrosis. Abnormal activated epithelial-mesenchymal transition (EMT) promotes the activation of hepatic stellate cells (HSCs). However, whether the antifibrotic effects of TQ occur through inhibiting EMT is largely unknown. In this study, it was found that TQ ameliorated liver fibrosis and collagen accumulation in carbon tetrachloride (CCl4) mice. In vitro, TQ inhibited HSC activation including reduced proliferation, α-smooth muscle actin, and collagen. In addition, TQ markedly suppressed the EMT process, with enhanced E-cadherin and reduced desmin. Notably, snail family transcriptional repressor 1 (Snai1), the EMT master transcription factor, was obviously inhibited by TQ in vivo and in vitro. Further studies demonstrated that Snai1 was a target of microRNA-30a (miR-30a), which was upregulated by TQ. Interestingly, the effects of TQ on HSC activation and EMT were almost inhibited by miR-30a inhibitor. Collectively, we demonstrate that TQ inhibits HSC activation, at least in part, via regulation of miR-30a and Snai1. TQ upregulates miR-30a expression, resulting in a reduced Snai1 level as well as EMT process inactivation, which contributes to the inhibition of HSC activation. TQ may be a potential therapeutic agent for liver fibrosis.
RESUMO
C-Jun N-terminal kinase (JNK) is a pivotal MAPK (mitogen-activated protein kinase), which activated by ischemia brain injury and plays a fairly crucial function in cerebral ischemic injury. Emerging studies demonstrated that JNK-IN-8 (a JNK inhibitor with high specificity) regulates traumatic brain injury through controlling neuronal apoptosis and inflammation. However, the function of JNK-IN-8 in ischemic stroke and the mechanisms underlying of JNK-IN-8 about neuroprotection are not well understood. In this work, male rats were treated with JNK-IN-8 after transient middle cerebral artery occlusion, and then the modified improved neurological function score (mNSS), the foot-fault test (FFT), interleukin-1ß (IL-1ß), IL-6, and tumor necrosis factor-α (TNF-α) levels were assessed. We found that JNK-IN-8-treated rats with MCAO exerted an observable melioration in space learning as tested by the improved mNSS, and showed sensorimotor functional recovery as measured by the FFT. JNK-IN-8 also played anti-inflammatory roles as indicated through decreased activation of microglia and decreased IL-6, IL-1ß, and TNF-α expression. Furthermore, JNK-IN-8 suppressed the activation of JNK and nuclear factor-κB (NF-κB) signaling as indicated by the decreased level of phosphorylated-JNK and p65. All data demonstrate that JNK-IN-8 inhibits neuroinflammation and improved neurological function by inhibiting JNK/NF-κB and is a promising agent for the prevention of ischemic brain injury.
Assuntos
Lesões Encefálicas Traumáticas/tratamento farmacológico , Hipóxia-Isquemia Encefálica/tratamento farmacológico , AVC Isquêmico/tratamento farmacológico , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Fármacos Neuroprotetores/farmacologia , Animais , Anti-Inflamatórios/farmacologia , Apoptose/efeitos dos fármacos , Lesões Encefálicas Traumáticas/patologia , Células Cultivadas , Hipóxia-Isquemia Encefálica/patologia , Inflamação/tratamento farmacológico , Interleucina-1beta/análise , Interleucina-6/análise , AVC Isquêmico/patologia , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Aprendizagem/efeitos dos fármacos , Masculino , Microglia/metabolismo , Artéria Cerebral Média/patologia , Neuroproteção/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Córtex Sensório-Motor/efeitos dos fármacos , Córtex Sensório-Motor/patologia , Fator de Transcrição RelA/metabolismo , Fator de Necrose Tumoral alfa/análiseRESUMO
Increasing studies have indicated that hypoxia serves as a pivotal microenvironmental factor that facilitates activation of hepatic stellate cells (HSCs). However, the mechanism by which hypoxia activates HSCs is not clear. Here, we demonstrated that plasmacytoma variant translocation 1 (PVT1) and autophagy were overexpressed in liver fibrotic specimens. In primary mouse HSCs, both PVT1 and autophagy were induced by hypoxia. Further study showed that hypoxia-induced autophagy depended on expression of PVT1 and miR-152 in HSCs. Luciferase reporter assay indicated that autophagy-related gene 14 (ATG14) was a direct target of miR-152. In addition, inhibition of autophagy by 3-methyladenine and Beclin-1 siRNA impeded activation of HSCs cultured in 1% O2. Taken together, autophagy induction via the PVT1-miR-152-ATG14 signaling pathway contributes to activation of HSCs under hypoxia condition.
Assuntos
Morte Celular Autofágica , Proteínas Relacionadas à Autofagia/metabolismo , Células Estreladas do Fígado/metabolismo , MicroRNAs/metabolismo , RNA Longo não Codificante/metabolismo , Transdução de Sinais , Proteínas de Transporte Vesicular/metabolismo , Animais , Hipóxia Celular , Células Estreladas do Fígado/patologia , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , CamundongosRESUMO
HOXA transcript at the distal tip (HOTTIP) has been shown to be up-regulated in a variety of cancers and is identified as an oncogenic long noncoding RNA. However, the biological role of HOTTIP in liver fibrosis is unclear. Here, we reported that HOTTIP was specifically overexpressed in activated hepatic stellate cells (HSCs). HOTTIP knockdown suppressed the activation and proliferation of HSCs. Luciferase reporter assay showed that HOTTIP and serum response factor (SRF) were targets of miR-150. RNA binding protein immunoprecipitation assay indicated the interaction between miR-150 and HOTTIP. Further study revealed that HOTTIP increased SRF expression as a competing endogenous RNA for miR-150, thus prompting HSC activation. Taken together, we provide a novel HOTTIP-miR-150-SRF signalling cascade in liver fibrosis.
Assuntos
Células Estreladas do Fígado/metabolismo , MicroRNAs/genética , RNA Longo não Codificante/genética , Fator de Resposta Sérica/genética , Animais , Carcinogênese/genética , Proliferação de Células/genética , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica/genética , Células Estreladas do Fígado/patologia , Humanos , Fígado/metabolismo , Fígado/patologia , Camundongos , Transdução de Sinais/genéticaRESUMO
Circular RNAs (circRNAs), often dysregulated in a variety of human diseases, participate in the initiation and development of cancers. Recently, circMTO1 (a circRNA derived from MTO1 gene), identified as a tumor suppressor, has been shown to contribute to the suppression of hepatocellular carcinoma. The present study aimed to explore the clinical significance and roles of circMTO1 in liver fibrosis. Here, we found that serum circMTO1 was significantly down-regulated in chronic hepatitis B (CHB) patients. Interestingly, serum circMTO1 was negatively correlated with fibrosis stages as well as HAI scores. Receiver operating characteristic curve analysis revealed that serum circMTO1 may serve as a diagnostic biomarker for liver fibrosis in CHB patients. Notably, overexpression of circMTO1 led to the suppression of transforming growth factor-ß1-induced hepatic stellate cells (HSCs) activation. Bioinformatic analysis and luciferase activity assays indicated that circMTO1 was a target of mircoRNA-17-5p (miR-17-5p). Data from RNA pull-down assay further confirmed that circMTO1 interacted with miR-17-5p. The inhibitory effects of circMTO1 on HSC activation were suppressed by miR-17-5p mimics. Further studies showed that Smad7 was a target of miR-17-5p. Moreover, circMTO1-inhibited HSC activation was also blocked down by loss of Smad7. Taken together, we demonstrate that circMTO1 inhibits liver fibrosis via regulation of miR-17-5p and Smad7, and serum circMTO1 may be a novel promising biomarker of liver fibrosis.
Assuntos
Cirrose Hepática/metabolismo , MicroRNAs/metabolismo , RNA Circular/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteína Smad7/metabolismo , Adulto , Carcinoma Hepatocelular/metabolismo , Estudos de Casos e Controles , Regulação para Baixo/fisiologia , Feminino , Células Estreladas do Fígado/metabolismo , Hepatite B Crônica/metabolismo , Humanos , Neoplasias Hepáticas/metabolismo , Masculino , Pessoa de Meia-Idade , Fator de Crescimento Transformador beta1/metabolismoRESUMO
BACKGROUND/AIMS: The activation of hepatic stellate cells (HSCs) is considered as a pivotal event in liver fibrosis and epithelial-mesenchymal transition (EMT) process has been reported to be involved in HSC activation. It is known that microRNAs (miRNAs) play a pro-fibrotic or anti-fibrotic role in HSC activation. Recently, emerging studies show that miR-30a is down-regulated in human cancers and over-expression of miR-30a inhibits tumor growth and invasion via suppressing EMT process. However, whether miR-30a could regulate EMT process in HSC activation is still unclear. METHODS: miR-30a expression was quantified using real-time PCR in carbon tetrachloride (CCl4)-induced rat liver fibrosis, activated HSCs and patients with cirrhosis. Roles of miR-30a in liver fibrosis in vivo and in vitro were also analyzed. Luciferase activity assays were performed to examine the binding of miR-30a to the 3'-untranslated region of snail family transcriptional repressor 1 (Snai1). RESULTS: miR-30a was down-regulated in human cirrhotic tissues. In CCl4 rats, reduced miR-30a was found in fibrotic liver tissues as well as isolated HSCs. There was a significant reduction in miR-30a in primary HSCs during culture days. miR-30a over-expression resulted in the suppression of CCl4-induced liver fibrosis. Restoration of miR-30a led to the inhibition of HSC activation including cell proliferation, α-SMA and collagen expression. Notably, miR-30a inhibited EMT process, with a reduction in TGF-ß1 and Vimentin as well as an increase in GFAP and E-cadherin. miR-30a induced a significant reduction in Snai1 protein expression when compared with the control. Interestingly, Snail protein expression was increased during liver fibrosis, indicating that there may be a negative correlation between miR-30a level and Snai1 protein expression. Further studies demonstrated that Snai1 was a target of miR-30a. CONCLUSION: Our results suggest that miR-30a inhibits EMT process, at least in part, via reduction of Snai1, leading to the suppression of HSC activation in liver fibrosis.
Assuntos
Transição Epitelial-Mesenquimal/fisiologia , Cirrose Hepática/patologia , MicroRNAs/metabolismo , Fatores de Transcrição da Família Snail/metabolismo , Regiões 3' não Traduzidas , Actinas/metabolismo , Animais , Antagomirs/metabolismo , Sequência de Bases , Caderinas/metabolismo , Tetracloreto de Carbono/toxicidade , Proliferação de Células , Células Cultivadas , Regulação para Baixo , Proteína Glial Fibrilar Ácida/genética , Proteína Glial Fibrilar Ácida/metabolismo , Células Estreladas do Fígado/citologia , Células Estreladas do Fígado/metabolismo , Humanos , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/genética , Masculino , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , Ratos , Ratos Sprague-Dawley , Alinhamento de Sequência , Fatores de Transcrição da Família Snail/antagonistas & inibidores , Fatores de Transcrição da Família Snail/genética , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismo , Vimentina/metabolismoRESUMO
BACKGROUND: 5-Fluorouracil (5-FU) has been widely applied to treat various types of cancers, including hepatocellular carcinoma (HCC). However, primary or acquired 5-FU resistance prevents the clinical application of this drug in cancer therapy. Herein, our study is the first to demonstrate that lower expression of KRAL, a long non-coding RNA (lncRNA), mediates 5-FU resistance in HCC via the miR-141/Keap1 axis. METHODS: Cell proliferation assays, western blot analysis, qRT-PCR, the dual-luciferase reporter assay and RNA immunoprecipitation were performed to investigate the mechanisms by which KRAL mediates 5-fluorouracil resistance in HCC cell lines. RESULTS: The quantitative analysis indicated that KRAL and Keap1 were significantly decreased and that Nrf2 was increased in HepG2/5-FU and SMMC-7721/5-FU cells compared with the corresponding expression levels in the respective parental cells. Overexpression of KRAL increased Keap1 expression, and inactivating the Nrf2-dependent antioxidant pathway could reverse the resistance of HepG2/5-FU and SMMC-7721/5-FU cells to 5-FU. Moreover, KRAL functioned as a competitive endogenous RNA (ceRNA) by effectively binding to the common miR-141 and then restoring Keap1 expression. These findings demonstrated that KRAL is an important regulator of Keap1; furthermore, the ceRNA network involving KRAL may serve as a treatment strategy against 5-FU resistance in hepatocellular carcinoma cells. CONCLUSIONS: KRAL/miR-141/Keap1 axis mediates 5-fluorouracil resistance in HCC cell lines.
Assuntos
Carcinoma Hepatocelular/patologia , Resistencia a Medicamentos Antineoplásicos/genética , Fluoruracila/farmacologia , Neoplasias Hepáticas/patologia , MicroRNAs/genética , RNA Longo não Codificante/genética , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , Técnicas de Silenciamento de Genes , Células Hep G2 , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Fator 2 Relacionado a NF-E2/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genéticaRESUMO
Homeobox transcript antisense RNA (HOTAIR), as a long intergenic non-coding RNA (lincRNA), is upregulated in various cancers and involved in diverse cellular functions. However, its role in liver fibrosis is unclear. In this study, HOTAIR expression was upregulated in hepatic stellate cells (HSCs) in vivo and in vitro during liver fibrosis. HOTAIR knockdown suppressed HSC activation including α-smooth muscle actin (α-SMA) and typeIcollagen in vitro and in vivo. Both HSC proliferation and cell cycle were inhibited by HOTAIR knockdown. Notably, inhibition of HOTAIR led to an increase in PTEN, associated with the loss of DNA methylation. miR-29b-mediated control of PTEN methylation was involved in the effects of HOTAIR knockdown. HOTAIR was confirmed a target of miR-29b and lack of the miR-29b binding site in HOTAIR prevented the suppression of miR-29b, suggesting HOTAIR contributes to PTEN expression downregulation via sponging miR-29b. Interestingly, increased HOTAIR was also observed in hepatocytes during liver fibrosis. Loss of HOTAIR additionally led to the increase in PTEN and the reduction in typeIcollagen in hepatocytes. Collectively, we demonstrate that HOTAIR downregulates miR-29b expression and attenuates its control on epigenetic regulation, leading to enhanced PTEN methylation, which contributes to the progression of liver fibrosis.
Assuntos
Epigênese Genética , Regulação da Expressão Gênica , Cirrose Hepática/genética , MicroRNAs/genética , PTEN Fosfo-Hidrolase/genética , RNA Longo não Codificante/genética , Regiões 3' não Traduzidas , Animais , DNA (Citosina-5-)-Metiltransferases/genética , Metilação de DNA , Modelos Animais de Doenças , Progressão da Doença , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Técnicas de Silenciamento de Genes , Células Estreladas do Fígado/metabolismo , Humanos , Cirrose Hepática/patologia , Camundongos , Modelos Biológicos , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-akt/metabolismo , DNA Metiltransferase 3BRESUMO
BACKGROUND/AIMS: It is known that the activation of hepatic stellate cells (HSCs) is a pivotal step in the initiation and progression of liver fibrosis. Aberrant activated Wnt/ß-catenin pathway is known to accelerate the development of liver fibrosis. microRNAs (miRNAs)-mediated Wnt/ß-catenin pathway has been reported to be involved in HSC activation during liver fibrosis. However, whether long noncoding RNAs (lncRNAs) regulate Wnt/ß-catenin pathway during HSC activation still remains unclear. METHODS: Long intergenic noncoding RNA-p21 (lincRNA-p21) expression was detected in Salvianolic acid B (Sal B)-treated cells. Effects of lincRNA-p21 knockdown on HSC activation and Wnt/ß-catenin pathway activity were analyzed in Sal B-treated cells. In lincRNA-p21-overexpressing cells, effects of miR-17-5p on HSC activation and Wnt/ß-catenin pathway activity were examined. RESULTS: LincRNA-p21 expression was up-regulated in HSCs after Sal B treatment. In primary HSCs, lincRNA-p21 expression was down-regulated at Day 5 relative to Day 2. Sal B-inhibited HSC activation including the reduction of cell proliferation, α-smooth muscle actin (α-SMA) and type I collagen was inhibited by lincRNA-p21 knockdown. Sal B-induced Wnt/ß-catenin pathway inactivation was blocked down by loss of lincRNA-p21. Notably, lincRNA-p21, confirmed as a target of miR-17-5p, suppresses miR-17-5p level. Lack of the miR-17-5p binding site in lincRNA-p21 prevents the suppression of miR-17-5p expression. In addition, the suppression of HSC activation and Wnt/ß-catenin pathway induced by lincRNA-p21 overexpression was almost inhibited by miR-17-5p. CONCLUSION: We demonstrate that lincRNA-p21-inhibited Wnt/ß-catenin pathway is involved in the effects of Sal B on HSC activation and lincRNA-p21 suppresses HSC activation, at least in part, via miR-17-5p-mediated-Wnt/ß-catenin pathway.
Assuntos
Benzofuranos/farmacologia , Células Estreladas do Fígado/metabolismo , MicroRNAs/metabolismo , RNA Longo não Codificante/metabolismo , Via de Sinalização Wnt/efeitos dos fármacos , beta Catenina/metabolismo , Animais , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Masculino , Camundongos , MicroRNAs/genética , RNA Longo não Codificante/genética , beta Catenina/genéticaRESUMO
BACKGROUND/AIMS: Recently, microRNAs (miRNAs) have been demonstrated to act as regulators of activation of hepatic stellate cells (HSCs). It is well known that the main profibrogenic inducer transforming growth factor-ß1 (TGF-ß1) contributes to HSC activation, which is a key event in liver fibrosis. Increasing studies show that miR-9-5p is down-regulated in liver fibrosis and restoration of miR-9-5p limits HSC activation. However, the role of miR-9-5p in TGF-ß1-induced HSC activation is still not clear. METHODS: miR-9-5p expression was quantified using real-time PCR in chronic hepatitis B (CHB) patients and TGF-ß1-treated LX-2 cells. In CHB patients, histological activity index (HAI) and fibrosis stages were assessed using the Ishak scoring system. Effects of miR-9-5p on liver fibrosis in vivo and in vitro were analyzed. Luciferase activity assays were performed to examine the binding of miR-9-5p to the 3'-untranslated region of type I TGF-ß receptor (TGFBR1) as well as TGFBR2. RESULTS: Compared with healthy controls, miR-9-5p was reduced in CHB patients. There was a lower miR-9-5p expression in CHB patients with higher fibrosis scores or HAI scores. miR-9-5p was down-regulated by TGF-ß1 in a dose-dependent manner. TGF-ß1-induced HSC activation including cell proliferation, α-SMA and collagen expression was blocked down by miR-9-5p. Notably, miR-9-5p ameliorates carbon tetrachloride-induced liver fibrosis. As determined by luciferase activity assays, TGFBR1 and TGFBR2 were targets of miR-9-5p. Further studies demonstrated that miR-9-5p inhibited TGF-ß1/Smads pathway via TGFBR1 and TGFBR2. Interestingly, promoter methylation was responsible for miR-9-5p down-regulation in liver fibrosis. The relationship between miR-9-5p expression and methylation was confirmed in CHB patients and TGF-ß1-treated cells. CONCLUSION: Our results demonstrate that miR-9-5p could inhibit TGF-ß1-induced HSC activation through TGFBR1 and TGFBR2. Loss of miR-9-5p is associated with its methylation status in liver fibrosis.
Assuntos
MicroRNAs/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Regiões 3' não Traduzidas , Actinas/genética , Actinas/metabolismo , Adulto , Animais , Antagomirs/metabolismo , Sequência de Bases , Tetracloreto de Carbono/toxicidade , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Regulação para Baixo/efeitos dos fármacos , Feminino , Células Estreladas do Fígado/citologia , Células Estreladas do Fígado/metabolismo , Hepatite B Crônica/genética , Hepatite B Crônica/metabolismo , Hepatite B Crônica/patologia , Humanos , Fígado/metabolismo , Fígado/patologia , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/genética , Cirrose Hepática/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , Pessoa de Meia-Idade , Regiões Promotoras Genéticas , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Receptor do Fator de Crescimento Transformador beta Tipo I , Receptor do Fator de Crescimento Transformador beta Tipo II , Receptores de Fatores de Crescimento Transformadores beta/antagonistas & inibidores , Receptores de Fatores de Crescimento Transformadores beta/genética , Alinhamento de Sequência , Fator de Crescimento Transformador beta1/farmacologiaRESUMO
Serum long non-coding RNAs (lncRNAs) are emerging as promising biomarkers for various human diseases. The aim of this study was to investigate the feasibility of using serum long intergenic non-coding RNA-p21 (lincRNA-p21) as a biomarker for chronic hepatitis B patients. Serum lincRNA-p21 levels were quantified using real-time PCR in 417 CHB patients and 363 healthy controls. The promoter methylation level of lincRNA-p21 was detected using bisulphite-sequencing analysis in primary hepatic stellate cells (HSCs). Sera from hepatitis B-infected patients contained lower levels of lincRNA-p21 than sera from healthy controls. Serum lincRNA-p21 levels negatively correlated with stages of liver fibrosis in infected patients. Receiver operating characteristic (ROC) curve analyses suggested that serum lincRNA-p21 had a significant diagnostic value for liver fibrosis in these patients. It yielded an area under the curve of ROC of 0.854 with 100% sensitivity and 70% specificity in discriminating liver fibrosis from healthy controls. There was additionally a negative correlation between serum lincRNA-p21 level and the markers of liver fibrosis including α-SMA and Col1A1. However, there was no correlation of serum lincRNA-p21 level with the markers of viral replication, liver inflammatory activity, and liver function. Notably, during primary HSCs culture, loss of lincRNA-p21 expression was associated with promoter methylation. Serum lincRNA-p21 could serve as a potential biomarker of liver fibrosis in CHB patients. Down-regulation of lincRNA-p21 in liver fibrosis may be associated with promoter methylation.
Assuntos
Biomarcadores/sangue , Inibidor de Quinase Dependente de Ciclina p21/genética , Hepatite B Crônica/complicações , Cirrose Hepática/diagnóstico , RNA Longo não Codificante/sangue , Soro/química , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Effective control of hepatic stellate cell (HSC) activation and proliferation is critical to the treatment of liver fibrosis. Long non-coding RNAs have been shown to play a pivotal role in the regulation of cellular processes. It has been reported that growth arrest-specific transcript 5 (GAS5) acts as a crucial mediator in the control of cell proliferation and growth. However, little is known about the role and underlying mechanism of GAS5 in liver fibrosis. In this study, our results indicated that GAS5 expression was reduced in mouse, rat, and human fibrotic liver samples and in activated HSCs. Overexpression of GAS5 suppressed the activation of primary HSCs in vitro and alleviated the accumulation of collagen in fibrotic liver tissues in vivo. We identified GAS5 as a target of microRNA-222 (miR-222) and showed that miR-222 could inhibit the expression of GAS5. Interestingly, GAS5 could also repress miR-222 expression. A pulldown assay further validated that GAS5 could directly bind to miR-222. As a competing endogenous RNAs, GAS5 had no effect on primary miR-222 expression. In addition, GAS5 was mainly localized in the cytoplasm. Quantitative RT-PCR further demonstrated that the copy numbers of GAS5 per cell are higher than those of miR-222. GAS5 increased the level of p27 protein by functioning as a competing endogenous RNA for miR-222, thereby inhibiting the activation and proliferation of HSCs. Taken together, a new regulatory circuitry in liver fibrosis has been identified in which RNAs cross-talk by competing for shared microRNAs. Our findings may provide a new therapeutic strategy for liver fibrosis.
Assuntos
Cirrose Hepática/fisiopatologia , RNA Longo não Codificante/fisiologia , RNA/genética , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
BACKGROUND/AIMS: Wnt/ß-catenin pathway is involved in liver fibrosis and microRNAs (miRNAs) are considered as key regulators of the activation of hepatic stellate cells (HSCs). A recent study showed the protective role of miR-378a-3p against cardiac fibrosis. However, whether miR-378a-3p suppresses Wnt/ß-catenin pathway in liver fibrosis is largely unknown. METHODS: miR-378a-3p expression was detected in carbon tetrachloride-induced liver fibrosis and activated HSCs. Effects of miR-378a-3p overexpression on HSC activation and Wnt/ß-catenin pathway were analyzed. Bioinformatic analysis was employed to identify the potential targets of miR-378a-3p. Serum miR-378a-3p expression was analyzed in patients with cirrhosis. RESULTS: Reduced miR-378a-3p expression was observed in the fibrotic liver tissues and activated HSCs. Up-regulation of miR-378a-3p inhibited HSC activation including cell proliferation, α-smooth muscle actin (α-SMA) and collagen expression. Moreover, miR-378a-3p overexpression resulted in Wnt/ß-catenin pathway inactivation. Luciferase reporter assays demonstrated that Wnt10a, a member of Wnt/ß-catenin pathway, was confirmed to be a target of miR-378a-3p. By contrast, miR-378a-3p inhibitor contributed to HSC activation, with an increase in cell proliferation, α-SMA and collagen expression. But all these effects were blocked down by silencing of Wnt10a. Notably, sera from patients with cirrhosis contained lower levels of miR-378a-3p than sera from healthy controls. Receiver operating characteristic curve analysis suggested that serum miR-378a-3p differentiated liver cirrhosis patients from healthy controls, with an area under the curve of ROC curve of 0.916. CONCLUSION: miR-378a-3p suppresses HSC activation, at least in part, via targeting of Wnt10a, supporting its potential utility as a novel therapeutic target for liver fibrosis.