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Recent studies suggest that circular RNA (circRNA)-mediated post-translational modification of RNA-binding proteins (RBP) plays a pivotal role in metastasis of hepatocellular carcinoma (HCC). However, the specific mechanism and potential clinical therapeutic significance remain vague. This study attempts to profile the regulatory networks of circRNA and RBP using a multi-omics approach. Has_circ_0006646 (circ0006646) is an unreported circRNA in HCC and is associated with a poor prognosis. Silencing of circ0006646 significantly hinders metastasis in vivo. Mechanistically, circ0006646 prevents the interaction between nucleolin (NCL) and the E3 ligase tripartite motif-containing 21 to reduce the proteasome-mediated degradation of NCL via K48-linked polyubiquitylation. Furthermore, the change of NCL expression is proven to affect the phosphorylation levels of multiple proteins and inhibit p53 translation. Moreover, patient-derived tumor xenograft and lentivirus injection, which is conducted to simulate clinical treatment confirmed the potential therapeutic value. Overall, this study describes the integrated multi-omics landscape of circRNA-mediated NCL ubiquitination degradation in HCC metastasis and provides a novel therapeutic target.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , ARN Circular , Ubiquitinación , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Humanos , ARN Circular/genética , ARN Circular/metabolismo , Ubiquitinación/genética , Ratones , Animales , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Línea Celular Tumoral , Nucleolina , Metástasis de la Neoplasia/genética , Proteínas de Motivos Tripartitos/genética , Proteínas de Motivos Tripartitos/metabolismo , Modelos Animales de Enfermedad , MultiómicaRESUMEN
Immunity-related GTPase M (IRGM), an Interferon-inducible protein, functions as a pivotal immunoregulator in multiple autoimmune diseases and infection. However, the role of IRGM in hepatocellular carcinoma (HCC) development remains unveiled. Here, we found interferon-γ (IFN-γ) treatment in HCC drastically triggered the expression of IRGM, and the high level of IRGM indicated poor prognosis in HCC patients. Functionally, IRGM promoted the malignant progression of HCC. Single-cell sequencing revealed that IRGM inhibition promoted the infiltration of CD8+ cytotoxic T lymphocytes (CTLs) with significant downregulation of PD-L1 expression in HCC. Furthermore, Immunoprecipitation-Mass Spectrometry assay revealed that IRGM interacted with transcription factor YBX1, which facilitated PD-L1 transcription. Mechanistically, IRGM promoted the interaction of YBX1 and phosphokinase S6K1, increasing phosphorylation and nuclear localization of YBX1, transcription of PD-L1. Additionally, the combination of IRGM inhibition with α-PD1 demonstrated a stronger anti-tumor effect compared to the single application of α-PD1. In summary, IRGM is a novel regulator of PD-L1, which suppresses CD8+ CTLs infiltration and function in HCC, resulting in cancer progression. This study may raise a novel therapeutic strategy combined with immune checkpoint inhibitors (ICIs) against HCC.
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Antineoplásicos , Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Antineoplásicos/uso terapéutico , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Carcinoma Hepatocelular/tratamiento farmacológico , Linfocitos T CD8-positivos , Proteínas de Unión al GTP/metabolismo , Neoplasias Hepáticas/tratamiento farmacológico , Fosforilación , Microambiente Tumoral , Proteína 1 de Unión a la Caja Y/genética , Proteína 1 de Unión a la Caja Y/metabolismo , Proteínas Quinasas S6 RibosómicasRESUMEN
In the course of antitumor therapy, the complex tumor microenvironment and drug-mediated changes in cell signaling and biological processes lead to drug resistance. The effect of sorafenib is greatly limited by the specific tumor microenvironment induced by antiangiogenic therapy and ferroptosis resistance induced by the upregulation of nuclear factor erythroid-2 related factor 2 (NRF2). In this study, a pH responsive and amphiphilic hyperbranched polyglycerol, HDP, is synthesized based on a co-graft click chemistry pathway. This nano-scale carrier provides excellent drug-loading capacity, storing stability and pH responsibility, and effectively co-delivery of sorafenib and siRNA. Sorafenib and siNRF2 plays a greatly synergistic effect in inducing reactive oxygen species (ROS), iron overloading, depleting glutathione (GSH), and promoting lipid peroxidation. Importantly, verified in two different animal experiments, HDP-ss (HDP loaded with both siNRF2 and sorafenib) presents a superior anti-tumor effect, by achieving a tumor inhibition rate of ≈94%. Thus, HDP can serve as an excellent targeted delivery nanocarrier with good biocompatibility in antitumor therapy, and combined application of siNRF2 effectively improves the antitumor effect of sorafenib by overcoming NRF2-mediated ferroptosis resistance. Taken together, this study provides a novel therapeutic strategy to combat the drug resistance in antiangiogenic therapy and ferroptosis.
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Carcinoma Hepatocelular , Ferroptosis , Neoplasias Hepáticas , Factor 2 Relacionado con NF-E2 , Sorafenib , Sorafenib/farmacología , Sorafenib/química , Ferroptosis/efectos de los fármacos , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/metabolismo , Humanos , Animales , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Portadores de Fármacos/química , Especies Reactivas de Oxígeno/metabolismo , Nanopartículas/química , Línea Celular Tumoral , Ratones , Glutatión/metabolismoRESUMEN
Ferroptosis, which is driven by iron-dependent lipid peroxidation, plays an essential role in liver ischemia-reperfusion injury (IRI) during liver transplantation (LT). Gp78, an E3 ligase, has been implicated in lipid metabolism and inflammation. However, its role in liver IRI and ferroptosis remains unknown. Here, hepatocyte-specific gp78 knockout (HKO) or overexpressed (OE) mice were generated to examine the effect of gp78 on liver IRI, and a multi-omics approach (transcriptomics, proteomics, and metabolomics) was performed to explore the potential mechanism. Gp78 expression decreased after reperfusion in LT patients and mice with IRI, and gp78 expression was positively correlated with liver damage. Gp78 absence from hepatocytes alleviated liver damage in mice with IRI, ameliorating inflammation. However, mice with hepatic gp78 overexpression showed the opposite phenotype. Mechanistically, gp78 overexpression disturbed lipid homeostasis, remodeling polyunsaturated fatty acid (PUFA) metabolism, causing oxidized lipids accumulation and ferroptosis, partly by promoting ACSL4 expression. Chemical inhibition of ferroptosis or ACSL4 abrogated the effects of gp78 on ferroptosis and liver IRI. Our findings reveal a role of gp78 in liver IRI pathogenesis and uncover a mechanism by which gp78 promotes hepatocyte ferroptosis by ACSL4, suggesting the gp78-ACSL4 axis as a feasible target for the treatment of IRI-associated liver damage.
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Ferroptosis , Hepatocitos , Hepatopatías , Receptores del Factor Autocrino de Motilidad , Daño por Reperfusión , Animales , Humanos , Ratones , Hepatocitos/enzimología , Inflamación/metabolismo , Hepatopatías/metabolismo , Daño por Reperfusión/metabolismo , Trasplante de Hígado , Receptores del Factor Autocrino de Motilidad/genética , Receptores del Factor Autocrino de Motilidad/metabolismo , Coenzima A LigasasRESUMEN
Hepatocellular carcinoma (HCC) is one of the most common malignancies with poor prognosis, and novel treatment strategies are urgently needed. Mitochondria are key regulators of cellular homeostasis and potential targets for tumor therapy. Here, the role of mitochondrial translocator protein (TSPO) in the regulation of ferroptosis and antitumor immunity is investigated and the potential therapeutic implications for HCC are assessed. TSPO is highly expressed in HCC and associated with poor prognosis. Gain- and loss-of-function experiments present that TSPO promotes HCC cell growth, migration, and invasion in vitro and in vivo. In addition, TSPO inhibits ferroptosis in HCC cells via enhancing the Nrf2-dependent antioxidant defense system. Mechanistically, TSPO directly interacts with P62 and interferes with autophagy, leading to the accumulation of P62. The P62 accumulation competes with KEAP1, preventing it from targeting Nrf2 for proteasomal degradation. Furthermore, TSPO promotes HCC immune escape by upregulating PD-L1 expression through Nrf2-mediated transcription. Notably, TSPO inhibitor PK11195 combines with anti-PD-1 antibody showing a synergistic anti-tumor effect in a mouse model. Overall, the results demonstrated that mitochondrial TSPO promotes HCC progression by inhibiting ferroptosis and antitumor immunity. Targeting TSPO can be a promising new strategy for HCC treatment.
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Carcinoma Hepatocelular , Ferroptosis , Neoplasias Hepáticas , Animales , Ratones , Carcinoma Hepatocelular/metabolismo , Proteínas Portadoras , Evasión Inmune , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/metabolismo , Proteínas Mitocondriales/metabolismo , Factor 2 Relacionado con NF-E2/metabolismoRESUMEN
BACKGROUND: Hepatocellular carcinoma (HCC) was one of the most prevalent life-threatening cancers. Metastasis is the leading cause of cancer-related death in HCC. MiRNAs play essential roles in cancer metastasis. METHODS: Expression of miR-652-3p in HCC was assessed. Function experiments of miR-652-3p and trinucleotide repeat-containing gene 6A protein (TNRC6A) were performed both in vitro and in vivo. mRNA sequencing, PCR, and western blot were performed to verify the target genes and pathway of miR-652-3p. The lung metastasis and xenograft cancer model in nude mice was established to investigate the effects of the miR-652-3p and TRNC6A on tumor metastasis in vivo. The relationship between the expression of the miR-652-3p, TNRC6A and the prognosis of HCC patients was analyzed. RESULTS: Upregulated miR-652-3p was found in the tumor tissues of HCC, especially in metastatic HCC patients. Overexpression of miR-652-3p promoted and knockdown of miR-652-3p suppressed HCC metastasis both in vitro and in vivo. MiR-652-3p promoted HCC metastasis via regulating the EMT pathway. TNRC6A was identified as a direct target of miR-652-3p, and the knockdown of TNRC6A restored repressed EMT and HCC metastasis caused by the inhibition of miR-652-3p. Clinical results revealed that high expression of miR-652-3p and low expression of TNRC6A were positively correlated to shortened overall survival and disease-free survival in HCC patients. CONCLUSIONS: MiR-652-3p promotes EMT and HCC metastasis by inhibiting TNRC6A expression in HCC. MiR-652-3p and TNRC6A may serve as potential biomarkers to predict prognosis in HCC patients with metastasis.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , MicroARNs , Animales , Humanos , Ratones , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Regulación Neoplásica de la Expresión Génica , Neoplasias Hepáticas/patología , Ratones Desnudos , MicroARNs/genética , MicroARNs/metabolismo , Metástasis de la NeoplasiaRESUMEN
The function of the N6-methyladenosine (m6A) methyltransferase RNA-binding motif protein 15 (RBM15) in hepatocellular carcinoma (HCC) has not been thoroughly investigated. Here we determined the clinical value, biological functions, and potential mechanisms of RBM15 in HCC. Expression of RBM15 was identified using tissue microarrays and online databases. A risk-prediction model based on RBM15 was developed and validated. We determined the biological role of RBM15 on HCC cells in vitro and in vivo. RNA sequencing was used to screen candidate targets of RBM15. Subsequently, the m6A dot blot assay, methylated RNA immunoprecipitation qPCR, dual-luciferase reporter assays, RNA decay assay, and RNA immunoprecipitation qPCR were employed to explore the mechanisms of RBM15. Our study showed that RBM15 was highly expressed in HCC, and overexpression of RBM15 indicated a worse outcome. A new nomogram combining RBM15 with age and TNM stage was developed and validated to predict the outcome of HCC patients; our nomogram increased the prediction accuracy of the TNM system. Functionally, RBM15 facilitates the proliferation and invasiveness of HCC. RBM15-mediated m6A modification contributed to a post-transcriptional activation of YES proto-oncogene 1 (YES1) in an insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1)-dependent manner. In addition, YES1 was confirmed as an oncogene in HCC cells by activating the mitogen-activated protein kinase (MAPK) pathway. In conclusion, RBM15-mediated m6A modification might facilitate the progression of HCC via the IGF2BP1-YES1-MAPK axis. RBM15 may be a promising biomarker in the outcome prediction of HCC.
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BACKGROUND: Mammalian Ste20-like kinase 4 (MST4), also known as serine/threonine kinase 26 (STK26), promotes development of several cancers and is found to be highly expressed in the placenta. However, in choriocarcinoma that originated from the placenta, the expression of MST4 was undetermined and its mechanism was unknown. In this study, the expression of MST4 in choriocarcinoma as well as the underlying mechanism was explored. PURPOSE: To detect the expression of MST4 in patient samples and mechanism of mediating EMT by MST4 in choriocarcinoma. PATIENTS AND METHODS: The metastatic lesions of choriocarcinoma (n=17) and volunteer villus (n=17) were collected to determine MST4 expression using immunohistochemistry and H&E staining. We use siRNA and lentiviral vector to knockdown MST4 and use plasmid to overexpress MST4 in choriocarcinoma. Then, we apply real-time polymerase chain reaction (RT-PCR), Western blot assay and immunofluorescence assay to detect target protein expressions. Cell invasion and migration and cell proliferation were detected by transwell assay and wound healing assay and CCK-8 and cell colony formation. RESULTS: MST4 is lowly expressed in the metastatic lesions of choriocarcinoma patients when compared with normal villus. Knockdown of MST4 activated epithelial-mesenchymal transition (EMT) process, significantly increasing the ability of invasion and migration in choriocarcinoma cell lines (JAR and JEG-3). In contrast, the EMT process was restrained in choriocarcinoma cell lines with overexpressed MST4. Meanwhile, genome-wide gene expression array, Western blot and ELISA revealed that tumor growth factor-beta 1 (TGF-ß1) has significantly increased. The EMT process and metastatic prompting biofunction were reversed after using TGF-ß1 inhibitor (LY364947) in the choriocarcinoma cell lines with MST4 knockdown. CONCLUSION: Our studies demonstrated that MST4 was lowly expressed in patient samples. Additionally, JAR and JEG-3 increase cell invasion and migration ability while there is no influence on cell proliferation with MST4 knockdown. Conversely, the metastatic ability of JAR and JEG-3 was decreased with overexpressed MST4. Moreover, TGF-ß1 was a key factor after MST4 knockdown. In conclusion, MST4 affects choriocarcinoma EMT by mediating TGF-ß1 expression.
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Exosomes play an important role in intercellular communication and metastatic progression of hepatocellular carcinoma (HCC). However, cellular communication between heterogeneous HCC cells with different metastatic potentials and the resultant cancer progression are not fully understood in HCC. Here, HCC cells with high-metastatic capacity (97hm and Huhm) were constructed by continually exerting selective pressure on primary HCC cells (MHCC-97H and Huh7). Through performing exosomal miRNA sequencing in HCC cells with different metastatic potentials (MHCC-97H and 97hm), many significantly different miRNA candidates were found. Among these miRNAs, miR-92a-3p was the most abundant miRNA in the exosomes of highly metastatic HCC cells. Exosomal miR92a-3p was also found enriched in the plasma of HCC patient-derived xenograft mice (PDX) model with high-metastatic potential. Exosomal miR-92a-3p promotes epithelial-mesenchymal transition (EMT) in recipient cancer cells via targeting PTEN and regulating its downstream Akt/Snail signaling. Furthermore, through mRNA sequencing in HCC cells with different metastatic potentials and predicting potential transcription factors of miR92a-3p, upregulated transcript factors E2F1 and c-Myc were found in high-metastatic HCC cells promote the expression of cellular and exosomal miR-92a-3p in HCC by directly binding the promoter of its host gene, miR17HG. Clinical data showed that a high plasma exosomal miR92a-3p level was correlated with shortened overall survival and disease-free survival, indicating poor prognosis in HCC patients. In conclusion, hepatoma-derived exosomal miR92a-3p plays a critical role in the EMT progression and promoting metastasis by inhibiting PTEN and activating Akt/Snail signaling. Exosomal miR92a-3p is a potential predictive biomarker for HCC metastasis, and this may provoke the development of novel therapeutic and preventing strategies against metastasis of HCC.
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Carcinoma Hepatocelular/genética , Exosomas/metabolismo , Neoplasias Hepáticas/genética , MicroARNs/metabolismo , Fosfohidrolasa PTEN/genética , Animales , Carcinoma Hepatocelular/sangre , Carcinoma Hepatocelular/mortalidad , Carcinoma Hepatocelular/secundario , Línea Celular Tumoral , Movimiento Celular/genética , Supervivencia sin Enfermedad , Factor de Transcripción E2F1/metabolismo , Transición Epitelial-Mesenquimal/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Hepáticas/sangre , Neoplasias Hepáticas/mortalidad , Neoplasias Hepáticas/patología , Masculino , Ratones , MicroARNs/genética , Persona de Mediana Edad , Metástasis de la Neoplasia/genética , Fosfohidrolasa PTEN/metabolismo , Pronóstico , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Transducción de Señal/genética , Regulación hacia Arriba , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: N6-methyladenosine (m6A) modification is an emerging layer of epigenetic regulation which is widely implicated in the tumorigenicity of hepatocellular carcinoma (HCC), offering a novel perspective for investigating molecular pathogenesis of this disease. The role of AlkB homolog 5 (ALKBH5), one of the m6A demethylases, has not been fully explored in HCC. Here we clarify the biological profile and potential mechanisms of ALKBH5 in HCC. METHODS: Expression of ALKBH5 and its correlation with clinicopathological characteristics of HCC were evaluated using tissue microarrays and online datasets. And biological effects of ALKBH5 in HCC were determined in vitro and in vivo. Subsequently, methylated RNA immunoprecipitation sequencing (MeRIP-seq) combined with RNA sequencing (RNA-seq), and following m6A dot blot, MeRIP-qPCR, RIP-qPCR or dual luciferase reporter assays were employed to screen and validate the candidate targets of ALKBH5. RESULTS: We demonstrated that ALKBH5 was down-regulated in HCC, and decreased ALKBH5 expression was an independent prognostic factor of worse survival in HCC patients. Functionally, ALKBH5 suppressed the proliferation and invasion capabilities of HCC cells in vitro and in vivo. Mechanistically, ALKBH5-mediated m6A demethylation led to a post-transcriptional inhibition of LY6/PLAUR Domain Containing 1 (LYPD1), which could be recognized and stabilized by the m6A effector IGF2BP1. In addition, we identified that LYPD1 induced oncogenic behaviors of tumors in contrast to ALKBH5. Dysregulation of ALKBH5/LYPD1 axis impelled the progression of HCC. CONCLUSION: Our study reveals that ALKBH5, characterized as a tumor suppressor, attenuates the expression of LYPD1 via an m6A-dependent manner in HCC cells. Our findings enrich the landscape of m6A-modulated tumor malignancy, and provide new insights into potential biomarkers and therapeutic targets of HCC treatment.
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Adenosina/análogos & derivados , Desmetilasa de ARN, Homólogo 5 de AlkB/metabolismo , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Epigénesis Genética , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Proteínas Supresoras de Tumor/genética , Adenosina/metabolismo , Animales , Carcinoma Hepatocelular/mortalidad , Carcinoma Hepatocelular/patología , Proteínas Portadoras , Línea Celular Tumoral , Proliferación Celular , Modelos Animales de Enfermedad , Proteínas Ligadas a GPI/genética , Regulación Neoplásica de la Expresión Génica , Xenoinjertos , Humanos , Neoplasias Hepáticas/patología , Ratones , Modelos Biológicos , Pronóstico , Unión Proteica , Transducción de SeñalRESUMEN
Hepatocellular carcinoma (HCC) is a highly heterogeneous, multigene-driven malignant tumor. Long chain acyl-CoA synthetase 4 (ACSL4), an enzyme has pivotal roles in arachidonic acid (AA) metabolism. However, its function and the underlying molecular mechanisms in HCC are still not fully elucidated. Here, we identified ACSL4 as a novel marker for AFP high subtype HCC through transcriptome profiling. ACSL4 was frequently upregulated in HCC samples and associated with poor prognosis. Functionally, ACSL4 knockdown resulted in decreased cell growth, whereas ectopic ACSL4 expression facilitated tumor formation in vitro and in vivo. Mechanistically, ACSL4 stabilized the oncoprotein c-Myc through ubiquitin-proteasome system in an ERK/FBW7-dependent manner. Cell growth ability mediated by ACSL4 elevation was partly attenuated by c-Myc depletion using siRNA or its inhibitor 10058-F4. In contrast, the effects of ACSL4 silencing were partially reversed by c-Myc overexpression via FBW7 knockdown. Clinically, ACSL4 expression was positively correlated with c-Myc in HCC. In conclusion, ACSL4 is a novel marker for AFP high subtype HCC. Our data uncovered a new mechanism by which ACSL4 promotes HCC progression via c-Myc stability mediated by ERK/FBW7/c-Myc axis and could be a valuable prognostic biomarker and a potential therapeutic target in HCC.
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Dimethyl fumarate (DMF) is generally used to treat psoriasis and multiple sclerosis. In the present study, we aimed to investigate the effects of DMF on hepatocellular carcinoma progression and its mechanism of action. In vitro, cell viability was examined using CCK-8 assay; cell cycle was analyzed by flow cytometry; angiogenesis was detected using tube formation assay; and autophagic flux assay results were examined using fluorescence microscopy. We also used western blotting to explore the potential mechanisms. In vivo, tumor xenograft experiment was performed with nude mice, and liver function, renal function, and routine blood counts were assessed using biochemical tests. Dimethyl fumarate inhibited tumor growth and angiogenesis in hepatocellular carcinoma, both in vitro and in vivo. Dimethyl fumarate decreased autophagy in hepatocellular carcinoma cells. Treatment with DMF activated SOCS3, which led to repression of JAK1 and STAT3 phosphorylation. DMF inhibited cell proliferation, angiogenesis, and autophagy via activation of the SOCS3/JAK1/STAT3 signaling pathway. This finding may provide a novel approach for the treatment of hepatocellular carcinoma.
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Liver cancer is a kind of lethal and aggressive malignant neoplasm with a high rate of relapse and metastasis after therapy. An important cause for the relapse and metastasis is the existence of liver cancer stem cells (CSCs), which have high resistance to chemotherapy and high tumorigenic potential. Therefore, it is crucial to develop new methods to eradicate CSCs in tumors. Herein, we develop a photodynamic therapy (PDT) that features bimodal metallacage-loaded nanoparticles (MNPs) for integrated chemotherapy. This platform achieves chemo-photodynamic combinational therapy. Organoplatinum(ii) metallacage-loaded nanoparticles show excellent ability to kill liver CSCs, decreasing their mobility and sphenoid formation ability under near-infrared laser irradiation. Importantly, MNPs can successfully penetrate into 3D tumor spheroids, which display higher drug resistance compared to traditional 2D cultured cells. This destroys CSCs and prevents subsequent tumor formation in vivo. With the excellent combinational therapeutic results in hand, the working mechanisms of MNPs were then studied. MNPs under NIR light irradiation can generate reactive oxygen species (ROS), resulting in damage of mitochondrial membrane and subsequent cell apoptosis with chemotherapeutic platinum. This study proves the great potential of MNPs for combinational cancer therapy, providing a new insight for the next generation of nanomedicines.
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Antineoplásicos/uso terapéutico , Neoplasias Hepáticas/tratamiento farmacológico , Nanopartículas del Metal/uso terapéutico , Células Madre Neoplásicas/efectos de los fármacos , Fármacos Fotosensibilizantes/uso terapéutico , Animales , Antineoplásicos/química , Antineoplásicos/efectos de la radiación , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Complejos de Coordinación/química , Complejos de Coordinación/efectos de la radiación , Complejos de Coordinación/uso terapéutico , Humanos , Rayos Infrarrojos , Hígado/patología , Masculino , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Nanopartículas del Metal/química , Nanopartículas del Metal/efectos de la radiación , Ratones Desnudos , Fotoquimioterapia , Fármacos Fotosensibilizantes/química , Fármacos Fotosensibilizantes/efectos de la radiación , Platino (Metal)/química , Platino (Metal)/efectos de la radiación , Porfirinas/química , Porfirinas/efectos de la radiación , Porfirinas/uso terapéutico , Especies Reactivas de Oxígeno/metabolismo , Esferoides Celulares/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: N6-methyladenosine (m6A) methylation, a well-known modification with new epigenetic functions, has been reported to participate in the tumorigenesis of hepatocellular carcinoma (HCC), providing novel insights into the molecular pathogenesis of this disease. However, as the key component of m6A methylation, Wilms tumor 1-associated protein (WTAP) has not been well studied in HCC. Here we investigated the biological role and underlying mechanism of WTAP in liver cancer. METHODS: We determined the expression of WTAP and its correlation with clinicopathological features using tissue microarrays and the Cancer Genome Atlas (TCGA) dataset. And we clarified the effects of WTAP on HCC cells using cell proliferation assay, colony formation, Edu assay and subcutaneous xenograft experiments. We then applied RNA sequencing combined with gene expression omnibus (GEO) data to screen candidate targets of WTAP. Finally, we investigated the regulatory mechanism of WTAP in HCC by m6A dot blot assay, methylated RNA immunoprecipitation (MeRIP) assay, dual luciferase reporter assay, RNA immunoprecipitation (RIP) assay and Chromatin immunoprecipitation (ChIP) assay. RESULTS: We demonstrated that WTAP was highly expressed in HCC which indicated the poor prognosis, and that WTAP expression served as an independent predictor of HCC survival. Functionally, WTAP promoted the proliferation capability and tumor growth of HCC cells in vitro and in vivo. Furthermore, ETS proto-oncogene 1 (ETS1) was identified as the downstream effector of WTAP. The m6A modification regulated by WTAP led to post-transcriptional suppression of ETS1, with the implication of Hu-Antigen R (HuR) as an RNA stabilizer. Then ETS1 was found to inhibit the progression of HCC and could rescue the phenotype induced by WTAP deficiency. Moreover, WTAP modulated the G2/M phase of HCC cells through a p21/p27-dependent pattern mediated by ETS1. CONCLUSION: We have identified that WTAP is significantly up-regulated in HCC and promotes liver cancer development. WTAP-guided m6A modification contributes to the progression of HCC via the HuR-ETS1-p21/p27 axis. Our study is the first to report that WTAP-mediated m6A methylation has a crucial role in HCC oncogenesis, and highlights WTAP as a potential therapeutic target of HCC treatment.
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Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteína 1 Similar a ELAV/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Metiltransferasas/metabolismo , Proteína Proto-Oncogénica c-ets-1/genética , Factores de Empalme de ARN/metabolismo , Animales , Carcinoma Hepatocelular/mortalidad , Carcinoma Hepatocelular/patología , Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral , Transformación Celular Neoplásica , Metilación de ADN , Modelos Animales de Enfermedad , Epigénesis Genética , Femenino , Silenciador del Gen , Humanos , Neoplasias Hepáticas/mortalidad , Neoplasias Hepáticas/patología , Masculino , Ratones , Modelos Biológicos , Estadificación de Neoplasias , Pronóstico , Proto-Oncogenes Mas , Factores de Empalme de ARN/genética , Carga Tumoral , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
MicroRNAs (miRNAs) have been validated to play prominent roles in the occurrence and development of many kinds of malignant cancer. MiR-424-5p has been reported to participate in various tumors proliferation and metastasis as a suppressor. On the contrary, miR-424-5p would promote cell proliferation in some tumors. However, the expression of miR-424-5p in intrahepatic cholangiocarcinoma (ICC) is rarely reported and its mechanism remains unclear. Here, we discover that miR-424-5p is frequently downregulated in ICC tissues compared with adjacent normal tissues and in ICC cells. Over-expression of miR-424-5p significantly inhibits the invasion and migration of ICC cells in vitro. Importantly, miR-424-5p is found to be a suppressor of ARK5, by binding to 3'-UTR of ARK5 mRNA and then inhibiting mTOR phosphorylated, thus deregulating epithelial-mesenchymal transition (EMT) of ICC. Furthermore, ARK5 is found to play a role in ICC metastasis and regulating EMT. Knockdown of ARK5 inhibits invasion and migration of ICC, while the over-expression gives an opposite effect. Besides, high-expression of ARK5 is also associated with poor prognosis. In conclusion, our study reveals that miR-424-5p is critical to the invasion, migration and EMT progression in ICC cells. Targeting the pathway described here may be a novel approach to inhibit metastasis of ICC and the restoration of miR-424-5p expression may be a promising strategy for ICC therapy.
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Movimiento Celular/fisiología , Proliferación Celular/fisiología , Supervivencia Celular/fisiología , Colangiocarcinoma/metabolismo , Neoplasias Hepáticas/metabolismo , MicroARNs/metabolismo , Proteínas Quinasas/metabolismo , Proteínas Represoras/metabolismo , Western Blotting , Línea Celular , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Supervivencia Celular/genética , Colangiocarcinoma/genética , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Neoplasias Hepáticas/genética , MicroARNs/genética , Proteínas Quinasas/genética , Proteínas Represoras/genéticaRESUMEN
BACKGROUND: Recently, increasing evidence has uncovered the roles of mRNA-miRNA-lncRNA network in multiple human cancers. However, a systematic mRNA-miRNA-lncRNA network linked to pancreatic cancer prognosis is still absent. METHODS: Differentially expressed genes (DEGs) were first identified by mining GSE16515 and GSE15471 datasets. DAVID database was utilized to conduct functional enrichment analysis. Protein-protein interaction (PPI) network was built using STRING database, and hub genes were identified by Cytoscape plug-in CytoHubba. Upstream miRNAs and lncRNAs of mRNAs were predicted by miRTarBase and miRNet, respectively. Expression, survival and correlation analysis for genes, miRNAs and lncRNAs were performed via GEPIA, Kaplan-Meier plotter and starBase. RESULTS: 734 and 180 upregulated and downregulated significant DEGs were identified, respectively. Functional enrichment analysis revealed that they were significantly enriched in focal adhesion, pathways in cancer and metabolic pathways. According to node degree, hub genes in the PPI networks were screened, such as TGFB1 and ALB. Among the top 20 hub genes, 7 upregulated genes and 2 downregulated hub genes had significant prognostic values in pancreatic cancer. 33 miRNAs were predicted to target the 9 key genes. But only high expression of 8 miRNAs indicated favorable prognosis in pancreatic cancer. Then, 90 lncRNAs were predicted to potentially bind to the 8 miRNAs. SCAMP1, HCP5, MAL2 and LINC00511 were finally identified as key lncRNAs. By combination of results from expression, survival and correlation analysis demonstrated that MMP9/ITGB1-miR-29b-3p-HCP5 competing endogenous RNA (ceRNA) sub-network was linked to prognosis of pancreatic cancer. CONCLUSIONS: In a word, we established a novel mRNA-miRNA-lncRNA sub-network, among which each RNA may be utilized as a prognostic biomarker of pancreatic cancer.
Asunto(s)
MicroARNs/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , ARN Largo no Codificante/metabolismo , ARN Mensajero/metabolismo , Bases de Datos Factuales , Regulación Neoplásica de la Expresión Génica , Redes Reguladoras de Genes , Predisposición Genética a la Enfermedad , Humanos , MicroARNs/genética , Neoplasias Pancreáticas/metabolismo , ARN Largo no Codificante/genética , ARN Mensajero/genética , Regulación hacia ArribaRESUMEN
Histone deacetylase inhibitors (HDACis) are the recommended treatment for many solid tumors; however, resistance is a major clinical obstacle for their efficacy. High levels of the transcription factor nuclear factor erythroid 2 like-2 (Nrf2) in cancer cells suggest a vital role in chemoresistance, and regulation of autophagy is one mechanism by which Nrf2 mediates chemoresistance. Although the molecular mechanisms underlying this activity are unclear, understanding them may ultimately improve therapeutic outcomes following HDACi treatment. In this study, we found that HDACi treatment increased Nrf2 mRNA and protein levels and enhanced Nrf2 transcriptional activity. Conversely, Nrf2 knockdown or inhibition blocked HDACi-induced autophagy. In addition, a microRNA (miRNA) array identified upregulation of miR-129-3p in response to Nrf2 overexpression. Chromatin immunoprecipitation assays confirmed miR-129-3p to be a direct Nrf2 target. RepTar and RNAhybrid databases indicated mammalian target of rapamycin (mTOR) as a potential miR-129-3p target, which we experimentally confirmed. Finally, Nrf2 inhibition or miR-129-3p in combination with HDACis increased cell death in vitro and in vivo. Collectively, these results demonstrated that Nrf2 regulates mTOR during HDACi-induced autophagy through miRNA-129-3p and inhibition of this pathway could enhance HDACi-mediated cell death.
Asunto(s)
MicroARNs/genética , Factor 2 Relacionado con NF-E2/genética , Neoplasias/tratamiento farmacológico , Serina-Treonina Quinasas TOR/genética , Animales , Autofagia/efectos de los fármacos , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Redes Reguladoras de Genes/genética , Xenoinjertos , Inhibidores de Histona Desacetilasas/farmacología , Humanos , Ratones , Factor 2 Relacionado con NF-E2/antagonistas & inhibidores , Neoplasias/genética , Neoplasias/patologíaRESUMEN
Intrahepatic cholangiocarcinoma (ICC) is a fatal, malignant tumor of the liver; effective diagnostic biomarkers and therapeutic targets for ICC have not been identified yet. High expression of H2A histone family member Z (H2A.Z) is a high-risk factor for poor prognosis in patients with breast cancer and primary hepatocellular cancer. However, the significance of H2A.Z and its expression in ICC remains unknown. The present study demonstrated that H2A.Z is overexpressed in ICC and expression of H2A.Z correlated with poor prognosis in patients with ICC. H2A.Z regulated cell proliferation in vitro and in vivo via H2A.Z/S-phase kinase-associated protein 2/p27/p21 signaling. Inhibition of H2A.Z reduced cell proliferation and induced apoptosis in ICC. In addition, downregulation of H2AZ reduced tumor metastasis by repressing epithelial-mesenchymal transition and enhanced the antitumor effects of cisplatin in the treatment of ICC. Overall, H2A.Z promoted cell proliferation and epithelial-mesenchymal transition in ICC, suggesting that H2A.Z may be a novel biomarker and therapeutic target for ICC.
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Neoplasias de los Conductos Biliares/tratamiento farmacológico , Neoplasias de los Conductos Biliares/metabolismo , Colangiocarcinoma/tratamiento farmacológico , Colangiocarcinoma/metabolismo , Cisplatino/farmacología , Histonas/biosíntesis , Animales , Antineoplásicos/farmacología , Neoplasias de los Conductos Biliares/genética , Neoplasias de los Conductos Biliares/patología , Biomarcadores de Tumor/biosíntesis , Biomarcadores de Tumor/genética , Carcinogénesis , Línea Celular Tumoral , Colangiocarcinoma/genética , Colangiocarcinoma/patología , Resistencia a Antineoplásicos , Xenoinjertos , Histonas/genética , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , PronósticoRESUMEN
The genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) screen is a powerful tool used to identify therapeutic targets that can be harnessed for cancer treatment. This study aimed to assess the efficacy of genome-wide CRISPR screening to identify druggable genes associated with sorafenib-treated hepatocellular carcinoma (HCC). A genome-scale CRISPR knockout (GeCKO v2) library containing 123,411 single guide RNAs (sgRNAs) was used to identify loss-of-function mutations conferring sorafenib resistance upon HCC cells. Resistance gene screens identified SGOL1 as an indicator of prognosis of patients treated with sorafenib. Of the 19,050 genes tested, the knockout screen identified inhibition of SGOL1 expression as the most-effective genetic suppressor of sorafenib activity. Analysis of the survival of 210 patients with HCC after hepatic resection revealed that high SGOL1 expression shortened overall survival (P = 0.021). Further, matched pairs analysis of the TCGA database revealed that SGOL1 is differentially expressed. When we used a lentivirus Cas9 vector to determine the effect of targeting SGOL1 with a specific sgRNA in HCC cells, we found that SGOL1 expression was efficiently inhibited and that loss of SGOL1 was associated with sorafenib resistance. Further, loss of SGOL1 from HCC cell decreased the cytotoxicity of sorafenib in vivo. We conclude that the CRISPR screen is a powerful tool for therapeutic target analysis of sorafenib treatment and that SGOL1 serves as a druggable target for HCC treated with sorafenib and an indicator of prognosis.
Asunto(s)
Antineoplásicos/farmacología , Carcinoma Hepatocelular/tratamiento farmacológico , Proteínas de Ciclo Celular/antagonistas & inhibidores , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Estudio de Asociación del Genoma Completo , Neoplasias Hepáticas/tratamiento farmacológico , Sorafenib/farmacología , Animales , Carcinoma Hepatocelular/mortalidad , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral , Resistencia a Antineoplásicos , Técnicas de Silenciamiento del Gen , Humanos , Neoplasias Hepáticas/mortalidad , Ratones , Ratones Endogámicos BALB C , Pronóstico , Sorafenib/uso terapéuticoRESUMEN
High porosities, large surface areas, and tunable functionalities made metal-organic frameworks (MOFs) as effective carriers for drug delivery. One of the most promising MOFs is the zeolitic imidazolate framework (ZIF-8) crystal, an advanced functional material for small-molecule delivery, due to its high loading ability and pH-sensitive degradation. As a novel carrier, ZIF-8 nanoparticles were used in this work to control the release of an autophagy inhibitor, 3-methyladenine (3-MA), and prevent it from dissipating in a large quantity before reaching the target. The cellular uptake in HeLa cells of 3-MA encapsulated in ZIF-8 (3-MA@ZIF-8 NPs) is facilitated through the nanoparticle internalization with reference to TEM observations and the quantitative analyses of zinc by ICP-MS. The autophagy-related proteins and autophagy flux in HeLa cells treated with 3-MA@ZIF-8 NPs show that the autophagosome formation is significantly blocked, which reveals that the pH-sensitive dissociation increases the efficiency of autophagy inhibition at the equivalent concentration of 3-MA. In vivo experiments, when compared to free 3-MA, 3-MA@ZIF-8 NPs show a higher antitumor efficacy and repress the expression of autophagy-related markers, Beclin 1 and LC3. It follows that ZIF-8 is an efficient drug delivery vehicle in antitumor therapy, especially in inhibiting autophagy of cancer cells.