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Background: The regulation of cancer stem cells (CSCs) is influenced by RNA-binding proteins (RBPs). The present study sought to investigate the role of NOVA2 in the processes of self-renewal, carcinogenesis, and lenvatinib resistance in liver CSCs. Methods: Neuro-oncological ventral antigen 2 (NOVA2) expression in liver CSCs was examined by real-time polymerase chain reaction (PCR). In vitro experiments were used to assess the effects of NOVA2 on liver CSC expansion and lenvatinib resistance. Results: In our study, the expression of the RBP NOVA2 was higher in CSCs. NOVA2 also increased the capacity for self-renewal and carcinogenesis of the liver CSCs via the Wnt pathway. Further, suppressing the Wnt pathway leads to desensitization of the hepatocellular carcinoma (HCC) cells that overexpress NOVA2 to apoptosis caused by lenvatinib. Analyzing patient data confirmed reduced levels of NOVA2 and therefore we speculate that NOVA2 may serve as a potential indicator for response to lenvatinib in patients with HCC. Methyltransferase-like 3 (METTL3) and YTH N6-methyladenosine RNA-binding protein 1 (YTHDF1)-dependent N6-methyladenosine (m6A) methylation were linked to upregulation of NOVA2 in HCC. Furthermore, it was shown that the expression of METTL3 was elevated in cellular models of type 2 diabetes mellitus (T2DM). Conclusions: NOVA2 is involved in the process of liver CSC self-renewal and carcinogenesis. In addition, NOVA2 expression may help identify patients with a higher chance of benefiting from lenvatinib treatment and can be a promising therapeutic target for HCC.
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BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) is a heterogeneous cancer with varying levels of liver tumor initiating or cancer stem cells in the tumors. We aimed to investigate the expression of different liver cancer stem cell (LCSC) markers in human HCCs and identify their regulatory mechanisms in stemness-related cells. METHODS: We used an unbiased, single-marker sorting approach by flow cytometry, fluorescence-activated cell sorting, and transcriptomic analyses on HCC patients' resected specimens. Knockdown approach was used, and relevant functional assays were conducted on the identified targets of interest. RESULTS: Flow cytometry on a total of 60 HCC resected specimens showed significant heterogeneity in the expression of LCSC markers, with CD24, CD13, and EpCAM mainly contributing to this heterogeneity. Concomitant expression of CD24, CD13, and EpCAM was detected in 32 HCC samples, and this was associated with advanced tumor stages. Transcriptomic sequencing on the HCC cells sorted for these individual markers identified epidermal growth factor receptor kinase substrate 8-like protein 3 (EPS8L3) as a common gene associated with the 3 markers and was functionally validated in HCC cells. Knocking down EPS8L3 suppressed the expression of all 3 markers. To search for the upstream regulation of EPS8L3, we found SP1 bound to EPS8L3 promoter to drive EPS8L3 expression. Furthermore, using Akt inhibitor MK2206, we showed that Akt signaling-driven SP1 drove the expression of the 3 LCSC markers. CONCLUSIONS: Our findings suggest that Akt signaling-driven SP1 promotes EPS8L3 expression, which is critical in maintaining the downstream expression of CD24, CD13, and EpCAM. The findings provide insight into potential LCSC-targeting therapeutic strategies.
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Antígeno CD24 , Carcinoma Hepatocelular , Molécula de Adhesión Celular Epitelial , Neoplasias Hepáticas , Células Madre Neoplásicas , Humanos , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/genética , Molécula de Adhesión Celular Epitelial/metabolismo , Molécula de Adhesión Celular Epitelial/genética , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/genética , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Antígeno CD24/metabolismo , Antígeno CD24/genética , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Biomarcadores de Tumor/metabolismo , Biomarcadores de Tumor/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Citometría de Flujo , Masculino , Femenino , Persona de Mediana EdadRESUMEN
BACKGROUND: Centrosome amplification is a well-recognized oncogenic driver of tumor initiation and progression across a variety of malignancies and has been linked with tumor aggressiveness, metastasis, and adverse prognosis. Nevertheless, the significance of centrosome amplification in HCC is not well understood. METHODS: The TCGA dataset was downloaded for centrosome amplification-related signature construction using the LASSO-penalized Cox regression algorithm, while the ICGC dataset was obtained for signature validation. Single-cell RNA sequencing from GSE149614 was analyzed to profile gene expression and the liver tumor niche. RESULTS: A total of 134 centrosome amplification-related prognostic genes in HCC were detected and 6 key prognostic genes (SSX2IP, SPAG4, SAC3D1, NPM1, CSNK1D, and CEP55) among them were screened out to construct a signature with both high sensitivity and specificity in diagnosis and prognosis of HCC patients. The signature, as an independent factor, was associated with frequent recurrences, high mortality rates, advanced clinicopathologic features, and high vascular invasions. Moreover, the signature was intimately associated with cell cycle-related pathways and TP53 mutation profile, suggesting its underlying role in accelerating cell cycle progression and leading to liver cancer development. Meanwhile, the signature was also closely correlated with immunosuppressive cell infiltration and immune checkpoint expression, making it a vital immunosuppressive factor in the tumor microenvironment. Upon single-cell RNA sequencing, SSX2IP and SAC3D1 were found to be specially expressed in liver cancer stem-like cells, where they promoted cell cycle progression and hypoxia. CONCLUSIONS: This study provided a direct molecular link of centrosome amplification with clinical characteristics, tumor microenvironment, and clinical drug-response, highlighting the critical role of centrosome amplification in liver cancer development and therapy resistance, thereby providing valuable insights into prognostic prediction and therapeutic response of HCC.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Pronóstico , Análisis de la Célula Individual , Centrosoma , Microambiente Tumoral/genética , Proteínas Asociadas a Microtúbulos , Proteínas de Ciclo CelularRESUMEN
Hepatocellular carcinoma (HCC), featured with high prevalence and poor prognosis, is the major cause of cancer-related deaths worldwide. As a subgroup of liver cancer cells capable of differentiation, tumorigenesis and self-renewal, liver cancer stem cells (LCSCs) serve as one of the reasons leading to HCC progression and therapeutic resistance. Therefore, in-depth exploration of novel molecular biomarkers related to LSCSs is of great necessity. In our study, we found that human AlkB homolog H5 (ALKBH5) expression was enriched in LCSCs, which could foster proliferation, invasion and migration of the HCC cells. Mechanically, ALKBH5 positively mediated the expression of SOX4 via demethylation, and SOX4 promoted SHH expression at the transcriptional level to activate sonic hedgehog (SHH) signaling pathway. Furthermore, exosomes derived from CD133+ HCC cells could transmit ALKBH5 into THP-1 cells, which might be associated with M2 polarization of macrophages. In summary, the ALKBH5/SOX4 axis plays a significant role in exacerbating LCSC properties via activating SHH signaling pathway, and ALKBH5 could be a critical effector related to macrophage M2 polarization. These findings might provide a promising new biomarker for HCC diagnosis and treatment.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/patología , Neoplasias Hepáticas/patología , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Transducción de Señal , Células Madre Neoplásicas/metabolismo , Factores de Transcripción SOXC/genética , Factores de Transcripción SOXC/metabolismo , Desmetilasa de ARN, Homólogo 5 de AlkB/metabolismoRESUMEN
Liver cancer stem cells (LCSCs) contribute to the initiation, metastasis, treatment resistance, and recurrence of hepatocellular carcinoma (HCC). Therefore, exploring potential anticancer agents targeting LCSCs may offer new therapeutic options to overcome HCC treatment failure. Hovenia dulcis Thunberg (HDT), a tree from the buckthorn family found in Asia, exhibits various biological activities, including antifatigue, antidiabetic, neuroprotective, hepatoprotective, and antitumor activities. However, the therapeutic effect of HDT in eliminating LCSCs remains to be confirmed. In this study, we evaluated the inhibitory activity of ethanol, chloroform, and ethyl acetate extracts from HDT branches on the growth of Huh7-derived LCSCs. The ethyl acetate extract of HDT (EAHDT) exhibited the most potent inhibitory activity against the growth of Huh7 LCSCs among the three HDT extracts. EAHDT suppressed the in vitro self-renewal ability of Huh7 LCSCs and reduced tumor growth in vivo using the Huh7 LCSC-transplanted chick embryo chorioallantoic membrane model. Furthermore, EAHDT not only arrested the cell cycle in the G0/G1 phase but also induced receptor-interacting protein kinase 3/mixed-lineage kinase domain-like protein-mediated necroptosis and caspase-dependent apoptosis in Huh7 LCSCs in a concentration-dependent manner. Furthermore, the growth inhibitory effect of EAHDT on Huh7 LCSCs was associated with the downregulation of c-MET-mediated downstream signaling pathways and key cancer stemness markers. Based on these findings, we propose that EAHDT can be used as a new natural drug candidate to prevent and treat HCC by eradicating LCSCs.
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Acetatos , Carcinoma Hepatocelular , Neoplasias Hepáticas , Embrión de Pollo , Animales , Necroptosis , Neoplasias Hepáticas/tratamiento farmacológico , Células Madre Neoplásicas , ApoptosisRESUMEN
A hepatocellular carcinoma tissue has mechanical heterogeneity, where the stiffness gradually increases from the core to the invasion front. Furthermore, there is evidence that stem cells from liver cancer (LCSCs) preferentially enrich the invasion front, exhibiting the stiffest modulus in the tumor. LCSCs have the features of stem/progenitor cells and play a vital part in liver cancer development. However, whether matrix stiffness affects LCSC stemness remains unclear. Here, we established a three-dimensional hydrogel for culturing LCSCs to simulate the stiffness of the core and the invasion front of a liver cancer tissue. The results showed that a stiffer matrix (72.2 ± 0.90 kPa) significantly potentiated LCSC stemness as compared with a soft matrix (7.7 ± 0.41 kPa). Moreover, Yes-associated protein signaling might mediate this promotion. Together, our findings illustrate the relationship between matrix stiffness and LCSC stemness, which may aid the production of novel treatment approaches against liver cancer.
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Carcinoma Hepatocelular , Proteínas Señalizadoras YAP , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Humanos , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patologíaRESUMEN
BackgroundPrimary liver cancer cells (PLCs) could more directly simulate the human tumor microenvironment. Compared with liver cancer cell lines, PLCs could reflect the human situation. As in previous studies, tumor stem cells were a small number of cancer cells in the microenvironment and considered to be one of the origins of liver cancer. This study aimed to screen stem cells in PLCs, analyze their biological characteristics, propose the possibility that liver cancer originated from stem cells.MethodsLiver cancer tissues of 17 patients were taken from the Affiliated Hospital of Guangdong Medical College, and PLCs were isolated by tissue slice method. The proliferation, tumor formation in nude mice, stem protein expression of PLCs were observed. C-kit+ liver cancer cells were screened and their biological characteristics were analyzed.ResultsPLCs could be stably passaged. Transmission electron microscopy indicated that the nucleus was irregular, there were many mitochondria, and the endoplasmic reticulum was irregularly distributed. PLCs could express E-Cadherin, Oct-4, β-Catenin, Sox2, CD326, C-kit, GPC3, Nanog. The proliferation curve of PLCs and Hep3B cells were similar, and they all could form tumors in nude mice. Flow-sorted C-kit+ PLCs, as well as C-kit+ Hep3B cells could highly express Bmi1, Sox2, Oct4, Notch1, Nanog, C-kit, β-Catenin, Smo, Nestin, ABCG2, ABCB1. And they also could clone and form tumors in vivo. But C-kit+ PLCs were more sensitive to chemotherapy drugs than C-kit+ liver cancer cell lines.ConclusionC-kit+ PLCs had the characteristics of tumor stem cells and were more sensitive to chemotherapy drugs.
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Humanos , Ciencias de la Salud , Neoplasias Hepáticas , Células Madre , Cultivo Primario de Células , Microscopía Electrónica , Células/inmunologíaRESUMEN
BACKGROUND: Primary liver cancer cells (PLCs) could more directly simulate the human tumor microenvironment. Compared with liver cancer cell lines, PLCs could reflect the human situation. As in previous studies, tumor stem cells were a small number of cancer cells in the microenvironment and considered to be one of the origins of liver cancer. This study aimed to screen stem cells in PLCs, analyze their biological characteristics, propose the possibility that liver cancer originated from stem cells. METHODS: Liver cancer tissues of 17 patients were taken from the Affiliated Hospital of Guangdong Medical College, and PLCs were isolated by tissue slice method. The proliferation, tumor formation in nude mice, stem protein expression of PLCs were observed. C-kit+ liver cancer cells were screened and their biological characteristics were analyzed. RESULTS: PLCs could be stably passaged. Transmission electron microscopy indicated that the nucleus was irregular, there were many mitochondria, and the endoplasmic reticulum was irregularly distributed. PLCs could express E-Cadherin, Oct-4, ß-Catenin, Sox2, CD326, C-kit, GPC3, Nanog. The proliferation curve of PLCs and Hep3B cells were similar, and they all could form tumors in nude mice. Flow-sorted C-kit+ PLCs, as well as C-kit+ Hep3B cells could highly express Bmi1, Sox2, Oct4, Notch1, Nanog, C-kit, ß-Catenin, Smo, Nestin, ABCG2, ABCB1. And they also could clone and form tumors in vivo. But C-kit+ PLCs were more sensitive to chemotherapy drugs than C-kit+ liver cancer cell lines. CONCLUSION: C-kit+ PLCs had the characteristics of tumor stem cells and were more sensitive to chemotherapy drugs.
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Neoplasias Hepáticas/patología , Células Madre Neoplásicas/patología , Animales , Línea Celular Tumoral , Humanos , Ratones , Ratones Desnudos , Células Tumorales CultivadasRESUMEN
BACKGROUND: Asymmetrical cell division (ACD) maintains the proper number of stem cells to ensure self-renewal. The rate of symmetric division increases as more cancer stem cells (CSCs) become malignant; however, the signaling pathway network involved in CSC division remains elusive. FXR (Farnesoid X receptor), a ligand-activated transcription factor, has several anti-tumor effects and has been shown to target CSCs. Here, we aimed at evaluating the role of FXR in the regulation of the cell division of CSCs. METHODS: The FXR target gene and downstream molecular mechanisms were confirmed by qRT-PCR, Western blot, luciferase reporter assay, EMAS, Chip, and IF analyses. Pulse-chase BrdU labeling and paired-cell experiments were used to detect the cell division of liver CSCs. Gain- and loss-of-function experiments in Huh7 cells and mouse models were performed to support findings and elucidate the function and underlying mechanisms of FXR-Notch1 in liver CSC division. RESULTS: We demonstrated that activation of Notch1 was significantly elevated in the livers of hepatocellular carcinoma (HCC) in Farnesoid X receptor-knockout (FXR-KO) mice and that FXR expression negatively correlated with Notch1 level during chronic liver injury. Activation of FXR induced the asymmetric divisions of Sox9+ liver CSCs and ameliorated liver injury. Mechanistically, FXR directs Sox9+ liver CSCs from symmetry to asymmetry via inhibition of Notch1 expression and activity. Deletion of FXR signaling or over-expression of Notch1 greatly increased Notch1 expression and activity along with ACD reduction. FXR inhibited Notch1 expression by directly binding to its promoter FXRE. FXR also positively regulated Numb expression, contributing to a feedback circuit, which decreased Notch1 activity and directed ACD. CONCLUSION: Our findings suggest that FXR represses Notch1 expression and directs ACD of Sox9+ cells to prevent the development of liver cancer.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Animales , División Celular Asimétrica , Carcinoma Hepatocelular/genética , Hígado , Neoplasias Hepáticas/genética , Ratones , Células Madre NeoplásicasRESUMEN
Long non-coding RNAs (lncRNAs) are important biological mediators that regulate numerous cellular processes. New experimental evidence suggests that lncRNAs play essential roles in liver development, normal liver physiology, fibrosis, and malignancy, including hepatocellular carcinoma and cholangiocarcinoma. In this review, we summarise our current understanding of the function of lncRNAs in the liver in both health and disease, as well as discuss approaches that could be used to target these non-coding transcripts for therapeutic purposes.
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BACKGROUND: MEG3 downregulated the expression in several tumors and inhibits human tumorigenesis. But so far, the mechanism of MEG3 in tumorigenesis is still unclear. METHODS: In gene infection, cellular and molecular technologies and tumorigenesis test in vitro and in vivo were performed, respectively. RESULTS: Our results indicate that MEG3 enhances the P53 expression by triggering the loading of P300 and RNA polymerase II onto its promoter regions dependent on HP1α. Moreover, MEG3 increases the methylation modification of histone H3 at the 27th lysine via P53. Furthermore, MEG3 inhibits the expression of TERT by increasing the H3K27me3 in TERT promoter regions, thereby inhibiting the activity of telomerase by reducing the binding of TERT to TERC. Furthermore, MEG3 also increases the expression of TERRA; therefore, the interaction between TERC and TERT was competitively attenuated by increasing the interaction between TERRA and TERT, which inhibits the activity of telomerase in hLCSCs. Strikingly, MEG3 reduces the length of telomere by blocking the formation of complex maintaining telomere length (POT1-Exo1-TRF2-SNM1B) and decreasing the binding of the complex to telomere by increasing the interplay between P53 and HULC. Ultimately, MEG3 inhibits the growth of hLCSCs by reducing the activity of telomerase and attenuating telomeric repeat binding factor 2(TRF2). CONCLUSIONS: Our results demonstrates MEG3 inhibits the occurrence of human liver cancer by blocking telomere, and these findings provide an important insight into the prevention and treatment of human liver cancer.
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Neoplasias Hepáticas , ARN Largo no Codificante , Telomerasa , Homólogo de la Proteína Chromobox 5 , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Hepáticas/genética , Células Madre Neoplásicas/metabolismo , ARN Largo no Codificante/genética , Telomerasa/genética , Telomerasa/metabolismo , Telómero/genética , Telómero/metabolismoRESUMEN
Cancer stem cells (CSCs) are a small subset of cells that sit atop the hierarchical ladder in many cancer types. Liver CSCs have been associated with high chemoresistance and recurrence rates in hepatocellular carcinoma (HCC). However, as of yet, no satisfactorily effective liver CSC-targeted treatment is available, which drove us to design and investigate the efficacy of a liposome-based delivery system. Here, we introduce a redox-triggered dual-targeted liposome, CEP-LP@S/D, capable of co-delivering doxorubicin (Dox) and salinomycin (Sal) for the synergistic treatment of liver cancer. This system is based on the association of CD133- and EpCAM-targeted peptides to form Y-shaped CEP ligands that were anchored to the surface of the liposome and allowed the selective targeting of CD133+ EpCAM+ liver CSCs. After arriving to the CSCs, the CEP-LP@S/D liposome undergoes endocytosis to the cytoplasm, where a high concentration of glutathione (GSH) breaks its disulfide bonds, thereby degrading the liposome. This then induces a rapid release of Dox and Sal to synergistically inhibit tumor growth. Notably, this effect occurs through Dox-induced apoptosis and concurrent lysosomal iron sequestration by Sal. Interestingly, both in vitro and in vivo studies indicated that our GSH-responsive co-delivery system not only effectively enhanced CSC targeting but also eliminated the non-CSC faction, thereby exhibiting high antitumor efficacy. We believe that the smart liposome nanocarrier-based co-delivery system is a promising strategy to combat liver cancer, which may also lay the groundwork for more enhanced approaches to target other cancer types as well.
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Hepatocellular carcinoma (HCC) is a common cancer with high morbidity and mortality. Poorer differentiation status indicates worse prognosis of HCC patients. Regain of better differentiation status may improve the prognosis. Differentiation therapy for HCC is based on the fact that agents may reverse the dedifferentiation process from hepatocytes to HCC cells and thus improve tumor differentiation status. Reversal of progenitor-like property and restoration of hepatic characteristics are main objectives of HCC differentiation therapy. Comprehending the mechanisms of HCC dedifferentiation provides ideas for drug design. Diverse dysregulated molecules and signalings cooperatively cause HCC dedifferentiation. Dysregulation of liver enriched transcription factors, especially hepatocyte nuclear factor 4α, was a critical determinant of HCC dedifferentiation. Aberrant pivotal signaling molecules such as transforming factor-ß, ß-catenin and Yes-associated protein caused disordered signalings, which promoted HCC dedifferentiation. Loss of epithelial morphology during epithelial-mesenchymal transition (EMT) concurred with HCC dedifferentiation. Some EMT-related molecules exerted double-sided role in concurrently inducing EMT and HCC dedifferentiation. Besides, microRNAs (e.g. miR-122 and miR-148a) as well as some impressive proteins (i.e. KLF4, gankyrin and CHD1L) functioned in manipulating HCC differentiation status. Restoring normal expression levels of these molecules could induce HCC differentiation and inhibited malignant tumor behaviors. Based on the knowledge above, some agents have been found effective in lab, but need more data to support their reliability. Additionally, peretinoin as a potential drug is in progress of several phase III clinical trials. It's promising that differentiation therapy for HCC may be a part of options in future HCC treatment.
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OBJECTIVE: Chemoresistance is one of the major barriers in chemotherapy-based hepatocellular carcinoma (HCC) intervention. 5-Fluorouracil (5-Fu) is a widely used as an anticancer drug. Liver cancer stem cells (LCSCs) are considered the origin of tumor recurrence and resistance. CCND1 (Cyclin D1) plays an important role in tumorigenesis and metastasis in multiple cancers including HCC. Herein, this study was designed to explore the role of CCND1 in regulating LCSCs differentiation and 5-Fu resistance in HCC cells. METHODS: The CCND1 mRNA level was examined by qRT-PCR. The protein levels of γ-H2AX (a DNA damage marker) and RAD51 (a DNA repair protein) were examined by Western blot. CD133 was used as a LCSC marker and CD133+ cell percentage in HCC cells was detected by flow cytometry. RESULTS: CCND1 silencing decreased CD133+ cell percentage in HepG2 and SMMC-7721 cells. Furthermore, CCND1 silencing significantly increased protein level of γ-H2AX and decreased that of RAD51 under 5-Fu exposure. Moreover, CCND1 silencing enhanced the sensitivity of HepG2 and SMMC-7721 cells to 5-Fu, which was effectively abrogated by RAD51 upregulation. CONCLUSION: Collectively, CCND1 silencing suppresses LCSCs differentiation and overcomes 5-Fu resistance in HCC.
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Antineoplásicos/farmacología , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Diferenciación Celular/genética , Ciclina D1/genética , Ciclina D1/fisiología , Resistencia a Antineoplásicos/genética , Fluorouracilo/farmacología , Silenciador del Gen , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Células Madre Neoplásicas/patología , Técnicas de Cultivo de Célula , Células Hep G2 , Humanos , Recombinasa Rad51/genética , Recombinasa Rad51/metabolismo , Regulación hacia Arriba/genéticaRESUMEN
Liver cancer stem cells (CSCs) are involved in tumorigenesis, progression, drug resistance and recurrence of hepatocellular carcinoma (HCC). However, the underlying mechanism for the propagation of liver cancer stem cells was unclear. Herein, we observed miR-206 expression was reduced in both chemoresistant HCCs and recurrent HCCs from patients. A dramatically decrease of miR-206 was detected in cluster of differentiation 133 (CD133) or epithelial cell adhesion molecule (EpCAM)-positive liver CSCs and in CSC-enriched hepatoma spheres. Functional studies revealed that a forced expression of miR-206 inhibited liver CSCs expansion by suppressing the dedifferentiation of hepatoma cells and attenuating the self-renewal of liver CSCs. Mechanistically, bioinformatic and luciferase reporter analysis identified epidermal growth factor receptor (EGFR) as a direct target of miR-206. Moreover, miR-206 downregulated the expression of EGFR in liver CSCs. There was a significant inverse correlation between miR-206 and EGFR mRNA expression in HCC samples. Special EGFR inhibitor Gefitinib abolished the discrepancy in liver CSC proportion and the self-renewal capacity between miR-206 overexpression hepatoma cells and control cells, which further confirmed that EGFR was required in miR-206-inhibited liver CSCs expansion. Conclusion: miR-206 could suppress HCC cell dedifferentiation and liver CSCs expansion by targeting EGFR signaling.
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Carcinoma Hepatocelular/metabolismo , Autorrenovación de las Células/genética , Neoplasias Hepáticas/metabolismo , MicroARNs/metabolismo , Células Madre Neoplásicas/metabolismo , Transducción de Señal/genética , Apoptosis/genética , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Carcinogénesis/genética , Carcinoma Hepatocelular/patología , Desdiferenciación Celular/genética , Autorrenovación de las Células/efectos de los fármacos , Regulación hacia Abajo , Resistencia a Antineoplásicos/genética , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/genética , Receptores ErbB/metabolismo , Gefitinib/farmacología , Células Hep G2 , Humanos , Neoplasias Hepáticas/patología , MicroARNs/genética , TransfecciónRESUMEN
Liver cancer stem cells (LCSCs) are responsible for the carcinogenesis, recurrence, metastasis and chemoresistance of hepatocellular carcinoma (HCC). This study aimed to explore the role of CCND1 (Cyclin D1), an oncogene in HCC, in regulating LCSC differentiation and to elucidate whether autophagy was involved in this process. The CCND1 mRNA level was examined by qRT-PCR. The protein levels of autophagy markers (Becin-1 and LC3II) were examined by western blot to evaluate autophagy level. CD133 was used as a LCSC marker and CD133 + cell percentage in HCC cells was detected by flow cytometry. Immunohistochemistry staining was performed to detect CD133 expression in SMMC-7721 cell-implanted tumor sections. The results showed that CCND1 expression was higher in HCC cell lines than that in normal hepatocyte L02 cells. Furthermore, CCND1 overexpression increased CD133 + cell percentage and protein levels of Becin-1 and LC3II in HepG2 and SMMC-7721 HCC cells. In contrast, CCND1 silencing exerted the opposite effect. Moreover, treatment with the autophagy activator rapamycin effectively abrogated the CCND1 silencing-reduced CD133 + cell percentage. Further in vivo assays demonstrated that CCND1 silencing decreased expression of CD133, Becin-1 and LC3II in xenograft tumors of SMMC-7721 cells. Taken together, CCND1 silencing suppresses LCSC differentiation through inhibiting autophagy.
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Autofagia/genética , Diferenciación Celular/genética , Ciclina D1/genética , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Células Madre/patologíaRESUMEN
Liver cancer stem cells contribute to tumorigenesis, progression, recurrence and drug resistance of hepatocellular carcinoma (HCC). However, the underlying mechanism for the propagation of liverCSCs is not fully understood yet. Here we show that miR-219 is upregulated in liver CSCs. Knockdown of miR-219 attenuates the self-renewal and tumorigenicity of liver CSCs. Conversely, miR-219 overexpressing enhances the self-renewal and tumorigenicity of liver CSCs.Mechanistically,miR-219 downregulates E-cadherin via itsmRNA 3'UTR in liver CSCs. The correlation between miR-219 and E-cadherin is validated in human HCC tissues. Furthermore, the miR-219 expression determines the responses of hepatoma cells to sorafenib treatment. Our findings indicate that miR-219 plays a critical role in liver CSCs expansion and sorafenib response, rendering miR-219 as an optimal target for the prevention and intervention of HCC.Abbreviations: HCC: Hepatocellular carcinoma; CSCs: cancer stem cells; DMEM: Dulbecco's modified Eagle's medium; FBS: fetal bovine serum; OS: overall survival.
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Cadherinas/genética , Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , MicroARNs/genética , Animales , Carcinogénesis/genética , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Proliferación Celular/genética , Transformación Celular Neoplásica/genética , Supervivencia sin Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica , Xenoinjertos , Humanos , Hígado/metabolismo , Hígado/patología , Neoplasias Hepáticas/patología , Masculino , Ratones , Recurrencia Local de Neoplasia/genética , Recurrencia Local de Neoplasia/patología , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Pronóstico , Transducción de Señal/genéticaRESUMEN
Liver CSCs are a rare subpopulation of heterogenous liver cancer cells with self-renewal and differentiation properties, which has emerged as a promising therapeutic target. Compelling data shows that NK cells selectively eliminate human cancer derived CSCs like colorectal carcinoma, melanoma, and glioblastoma. But the effect of NK cells on liver CSCs still remains unknown. To study the cytotoxic effect of NK cells on liver CSCs and the mechanism, we performed cytotoxicity assay, ELISA assays, CRISPRi, qRT-PCR, immunoblotting, RNA immunoprecipitation, and luciferase reporter using two types of CSCs reprogrammed from HCC. CSCs derived from liver cancer were susceptible to NK cell mediated cytotoxicity. The susceptibility of liver CSCs to NK cell-mediated cytotoxicity declined significantly after silencing CD44 by CRISPRi-mediated gene knockdown. CD44 3' UTR functioned as a ceRNA to regulate the expression of ULBP2 mainly by competing miR-34a. CD44 3' UTR functioned as a ceRNA to enhance NK sensitivity of liver cancer stem cell by regulating ULBP2 expression.
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Carcinoma Hepatocelular/metabolismo , Receptores de Hialuranos/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Células Asesinas Naturales/metabolismo , Neoplasias Hepáticas/metabolismo , Carcinoma Hepatocelular/genética , Línea Celular , Ensayo de Inmunoadsorción Enzimática , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/metabolismo , Regulación Neoplásica de la Expresión Génica/genética , Regulación Neoplásica de la Expresión Génica/fisiología , Humanos , Receptores de Hialuranos/genética , Immunoblotting , Inmunoprecipitación , Péptidos y Proteínas de Señalización Intercelular/genética , Neoplasias Hepáticas/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
BACKGROUND: Liver cancer stem cells (LCSCs) are a small subset of cells characterized by unlimited self-renewal, cell differentiation, and uncontrollable cellular growth. LCSCs are also resistant to conventional therapies and are thus believed to be held responsible for causing treatment failure of hepatocellular carcinoma (HCC). It has been recently found that long non-coding RNAs (lncRNAs) are important regulators in HCC. This present study aims to explore the underlying mechanism of how lncRNA DLX6-AS1 influences the development of LCSCs and HCC. METHODS: A microarray-based analysis was performed to initially screen differentially expressed lncRNAs associated with HCC. We then analyzed the lncRNA DLX6-AS1 levels as well as CADM1 promoter methylation. The mRNA and protein expression of CADM1, STAT3, CD133, CD13, OCT-4, SOX2, and Nanog were then detected. We quantified our results by evaluating the spheroid formation, proliferation, and tumor formation abilities, as well as the proportion of tumor stem cells, and the recruitment of DNA methyltransferase (DNMT) in LCSCs when lncRNA DLX6-AS1 was either overexpressed or silenced. RESULTS: LncRNA DLX6-AS1 was upregulated in HCC. The silencing of lncRNA DLX6-AS1 was shown to reduce and inhibit spheroid formation, colony formation, proliferation, and tumor formation abilities, as well as attenuate CD133, CD13, OCT-4, SOX2, and Nanog expression in LCSCs. Furthermore, downregulation of lncRNA DLX6-AS1 contributed to a reduction in CADM1 promoter methylation via suppression of DNMT1, DNMT3a, and DNMT3b in LCSCs and inactivating the STAT3 signaling pathway. CONCLUSION: This study demonstrated that down-regulated lncRNA DLX6-AS1 may inhibit the stem cell properties of LCSCs through upregulation of CADM1 by suppressing the methylation of the CADM1 promoter and inactivation of the STAT3 signaling pathway.
Asunto(s)
Molécula 1 de Adhesión Celular/genética , Transformación Celular Neoplásica/genética , Metilación de ADN , Proteínas de Homeodominio/genética , Regiones Promotoras Genéticas , ARN Largo no Codificante/genética , Factor de Transcripción STAT3/metabolismo , Transducción de Señal , Animales , Biomarcadores de Tumor , Línea Celular Tumoral , Proliferación Celular , Transformación Celular Neoplásica/metabolismo , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Silenciador del Gen , Xenoinjertos , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Ratones , Modelos Biológicos , Células Madre Neoplásicas/metabolismoRESUMEN
Liver cancer stem cells (CSCs) contribute to tumorigenesis, progression, drug resistance and recurrence of hepatocellular carcinoma (HCC). However, the underlying mechanism for the propagation of liver CSCs remains unclear. Herein, we observed miR-613 expression was downregulated in both chemoresistant and recurrent HCC patients. A remarkable decrease in miR-613 was detected in CD24 or OV6-positive liver CSCs and CSC-enriched hepatoma spheres. Down-regulation of miR-613 facilitated liver CSCs expansion by promoting the dedifferentiation of hepatoma cells and enhancing the self-renewal of liver CSCs. Mechanistically, bioinformatic and luciferase reporter analysis identified SOX9 as a direct target of miR-613. Overexpression of miR-613 inhibited the expression of SOX9 in HCC cells. Special SOX9 siRNA abolished the discrepancy in liver CSCs proportion and the self-renewal capacity between miR-613 overexpression hepatoma cells and control cells, which further confirmed that SOX9 was required in miR-613-inhibited liver CSCs expansion. Furthermore, hepatoma cells with miR-613 overexpression performed more sensitivity to cisplatin or sorafenib treatment. Conclusion: miR-613 could inhibit HCC cell dedifferentiation and liver CSCs expansion by targeting SOX9 signaling and may prove to be a novel therapeutic target for HCC patients.