RESUMO
The prevention and early diagnosis of liver cancer remains a global medical challenge. During the malignant transformation of hepatocytes, a variety of oncogenic cellular signalling molecules, such as novel high mobility group-Box 3, angiopoietin-2, Golgi protein 73, glypican-3, Wnt3a (a signalling molecule in the Wnt/ß-catenin pathway), and secretory clusterin, can be expressed and secreted into the blood. These signalling molecules are derived from different signalling pathways and may not only participate in the malignant transformation of hepatocytes but also become early diagnostic indicators of hepatocarcinogenesis or specific targeted molecules for hepatocellular carcinoma therapy. This article reviews recent progress in the study of several signalling molecules as sensitive biomarkers for monitoring hepatocarcinogenesis.
RESUMO
[This corrects the article DOI: 10.3389/fimmu.2023.1183768.].
RESUMO
BACKGROUND: The effective treatment for hepatocellular carcinoma (HCC) depends on early diagnosis. Previously, the abnormal expression of Wnt3a as the key signaling molecule in the Wnt/ß-catenin pathway was found in HCC cells and could be released into the circulation. In this study, we used rat model of hepatocarcinogenesis to dynamically investigate the alteration of oncogenic Wnt3a and to explore its early monitor value for HCC. METHODS: Sprague-Dawley rats (SD) were fed with diet 2-fluorenylacetamide (2-FAA, 0.05%) for inducing hepatocarcinogenesis, and grouped based on liver morphological alteration by Hematoxylin & Eosin (H&E) staining; rats fed with normal chow were used as normal control (NC). Total RNA and protein were purified from rat livers. Differently expressed genes (DEGs) or Wnt3a mRNA, cellular distribution, and Wnt3a protein levels were analyzed by whole genome microarray with signal logarithm ratio (SLR log2cy5/cy3), immunohistochemistry, and enzyme-linked immunosorbent assay, respectively. RESULTS: Models of rat hepatocarcinogenesis were successfully established based on liver histopathological H&E staining. Rats were divided into the cell degeneration (rDeg), precancerosis (rPre-C) and HCC (rHCC) groups. Total numbers of the up- and down-regulated DEGs with SLR ≥ 8 were 55 and 48 in the rDeg group, 268 and 57 in the rPre-C group, and 312 and 201 in the rHCC group, respectively. Significantly altered genes were involved in cell proliferation, signal transduction, tumor metastasis, and apoptosis. Compared with the NC group, Wnt3a mRNA was increased by 4.6 folds (P < 0.001) in the rDeg group, 7.4 folds (P < 0.001) in the rPre-C group, and 10.4 folds (P < 0.001) in the rHCC group; the positive rates of liver Wnt3a were 66.7% (P = 0.001) in the rDeg group, 100% (P < 0.001) in the rPre-C group, and 100% (P < 0.001) in the rHCC group, respectively. Also, there were significant differences of liver Wnt3a (P < 0.001) or serum Wnt3a (P < 0.001) among different groups. CONCLUSIONS: Overexpression of Wnt3a was associated with rat hepatocarcinogenesis and it should be expected to be a promising monitoring biomarker for HCC occurrence at early stage.
Assuntos
Carcinogênese , Carcinoma Hepatocelular , Neoplasias Hepáticas , Proteína Wnt3A , Animais , Ratos , Biomarcadores Tumorais/metabolismo , Carcinogênese/genética , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/patologia , Ratos Sprague-Dawley , RNA Mensageiro/metabolismo , Via de Sinalização Wnt , Proteína Wnt3A/análiseRESUMO
BACKGROUND: Insulin-like growth factor-1 receptor (IGF-1R) is over-expressed in hepatocellular carcinoma (HCC). However, the relationship between IGF-1R activation and HCC progression remains unidentified. AIM: To investigate the effects of editing IGF-1R on the biological features of HCC cells. METHODS: Immunohistochemistry analyzed the expressions of IGF-1R and P-glyco protein (P-gp) in HCC tissues and their distal non-cancerous tissues (non-Ca). IGF-1R was edited with Crispr/Cas9 system, screened specific sgRNAs, and then transfected into HepG2 cells. CCK-8, scratch wound test detected cell proliferation, migration, invasion and transwell assays, respectively. Alterations of IGF-1R and P-gp were confirmed by Western blotting. Alterations of anti-cancer drug IC50 values were analyzed at the cell level. RESULTS: The positive rates of IGF-1R (93.6%, χ 2 = 63.947) or P-gp (88.2%, χ2 = 58.448) were significantly higher (P < 0.001) in the HCC group than those (36.6% in IGF-1R or 26.9% in P-gp) in the non-Ca group. They were positively correlated between high IGF-1R and P-gp expression, and they were associated with hepatitis B virus infection and vascular invasion of HCC. Abnormal expressions of circulating IGF-1R and P-gp were confirmed and associated with HCC progression. Biological feature alterations of HCC cells transfected with specific sgRNA showed IGF-1R expression down-regulation, cell proliferation inhibition, cell invasion or migration potential decreasing, and enhancing susceptibility of HepG2 cells to anti-cancer drugs. CONCLUSION: Edited oncogenic IGF-1R was useful to inhibit biological behaviors of HepG2 cells.
RESUMO
Acute kidney injury (AKI) is a common critical clinical disease characterized by a sharp decline of renal function. Ischemia-reperfusion (IR) is one of the main causes of AKI. The mortality of AKI remains high due to the lack of early diagnosis and cause specific treatment. IR rapidly initiates innate immune responses, activates complement and innate immune cells, releasing a large number of injury-related molecules such as high mobility group box-1 (HMGB1), inflammatory mediators such as caspase-3, and then recruits immune inflammatory cells including M1 macrophages (MÏ) to the microenvironment of injury, causing apoptosis and necrosis of renal tubular epithelial cells (TECs). Dead cells and associated inflammation further activate the adaptive immune system, which not only aggravates tissue damage, but also initiates M2 MÏ participated inflammatory clearance, tissue repair and regeneration. MÏ, professional phagocytes, and TECs, semi-professional phagocytes, can phagocytose around damaged cells including apoptotic MÏ and TECs, which are key innate immune cells to regulate the outcome of injury, repair or fibrosis. In recent years, it has been found that erythropoietin (EPO) not only binds to the homodimeric receptor (EPOR)2 to induce erythropoiesis, but also binds to the heterodimeric receptor EPOR/ßcR, also known as innate repair receptor, which plays renoprotective roles. Properdin is the only positive regulator in the complement activation of alternative pathway. It also can effectively identify and bind to early apoptotic T cells and facilitate phagocytic clearing by MÏ through a non-complement activation-dependent mechanism. Our previous studies have shown that MÏ and TECs associated with EPO and its receptors and properdin are involved in IR injury and repair, but the underlying mechanism needs to be further explored. As an important carrier of cell-to-cell signal transmission, exosomes participate in the occurrence and development of a variety of renal diseases. The role of exosomes involved in the interaction between MÏ and TECs in IR-induced AKI is not fully defined. Based on the available results in the role of MÏ and TECs in renal IR-induced AKI, this review discussed the role of MÏ polarization and interaction with TECs in renal IR injury, as well as the participation of EPO and its receptors, properdin and exosomes.
Assuntos
Injúria Renal Aguda , Traumatismo por Reperfusão , Injúria Renal Aguda/etiologia , Injúria Renal Aguda/metabolismo , Animais , Células Epiteliais/metabolismo , Humanos , Isquemia/metabolismo , Rim , Macrófagos/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , ReperfusãoRESUMO
BACKGROUND: Hepatic Golgi protein-73 (GP73) expression is related to hepatocellular carcinoma (HCC) progression. The aim of this study was to investigate the dynamic expression of GP73 mRNA and protein during hepatocytes malignant transformation. METHODS: Human GP73 expressions in 88 HCC tissues and their self-control surrounding tissues were examined by immunohistochemistry, and survival time of HCC patients was evaluated by the Kaplan-Meier method. HCC model of Sprague-Dawley rats was made by diet containing 2-fluorenylacetamide. The rats were divided into the control, hepatocyte degeneration, precanceration, and HCC groups to observe GP73 protein and mRNA alterations during hepatocytes malignant transformation. RESULTS: The GP73 expression was significantly higher in the cancerous tissues than that in the surrounding tissues, with shorter survival time, and the positive rates of GP73 protein in human HCC tissues were 53.3% at stage I, 84.0% at stage II, 84.6% at stage III, and 60.0% at stage IV, respectively. The positive rates of hepatic GP73 protein and mRNA in the rat models were none in the control group, 66.7% and 44.4% in the hepatocytes degeneration group, 88.9% and 77.8% in the hepatocytes precanceration group, and 100% in the HCC group, respectively. There was a positive correlation (r = 0.91, P<0.01) between hepatic GP73 and serum GP73 during rat hepatocytes malignant transformation. CONCLUSIONS: Abnormal GP73 expression may be a sensitive and valuable biomarker in hepatocarcinogensis.
Assuntos
Biomarcadores Tumorais/metabolismo , Carcinoma Hepatocelular/metabolismo , Transformação Celular Neoplásica/metabolismo , Hepatócitos/metabolismo , Neoplasias Hepáticas/metabolismo , Proteínas de Membrana/metabolismo , Adulto , Idoso , Animais , Biomarcadores Tumorais/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/mortalidade , Carcinoma Hepatocelular/patologia , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Feminino , Regulação Neoplásica da Expressão Gênica , Hepatócitos/patologia , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/mortalidade , Neoplasias Hepáticas/patologia , Masculino , Proteínas de Membrana/genética , Pessoa de Meia-Idade , Estadiamento de Neoplasias , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos Sprague-Dawley , Fatores de TempoRESUMO
BACKGROUND: Although secretory clusterin (sCLU) plays a crucial role in Hepatocellular Carcinoma (HCC) cells proliferation, Multiple Drug Resistance (MDR), metastasis and so on, its targeted effects and exact mechanism are still unknown. This review summarizes some new progress in sCLU as a molecular-targeted therapy in the treatment of HCC. METHODS: A systematic review of the published English-language literature about sCLU and HCC has been performed using the PubMed and bibliographic databases. Some valuable studies on sCLU in HCC progression were searched for relevant articles with the keywords: HCC, diagnosis, MDR, as molecular-targeted in treatment, and so on. RESULTS: The incidence of the positive rate of sCLU was significantly higher in HCC tissues as compared to the surrounding tissues at mRNA or protein level, gradually increasing with tumor-nodemetastasis staging (P<0.05). Also, the abnormal level of sCLU was related to poor differentiation degree, and considered as a useful marker for HCC diagnosis or independent prognosis for patients. Hepatic sCLU could be silenced at mRNA level by specific sCLU-shRNA or by OGX-011 to inhibit cancer cell proliferation with an increase in apoptosis, cell cycle arrest, reversal MDR, alteration of cell migration or invasion behaviors, and a decrease in GSK-3ß or AKT phosphorylation in vitro, as well as significant suppression of the xenograft growth by down-regulating ß-catenin, p-GSK3ß, and cyclinD1 expression in vivo. CONCLUSION: Abnormal hepatic sCLU expression should not only be a new diagnostic biomarker but also a novel promising target for inhibiting HCC growth.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Linhagem Celular Tumoral , Proliferação de Células , Clusterina , Glicogênio Sintase Quinase 3 beta , HumanosRESUMO
Secretory clusterin (sCLU) is associated with hepatocellular carcinoma (HCC) progression by contributing to angiogenesis, chemoresistance, cell survival, and metastasis. However, the sCLU expression at early stage of HCC progression remains to be clarified. In this study, the alteration of sCLU oncogenicity was firstly evaluated in HCC- and their para-cancerous- tissues. The incidence of sCLU expression in HCC was significantly higher than that in their non-tumorous tissues at message RNA (mRNA) or protein level, gradually increasing with tumor-node-metastasis (TNM) staging. Abnormal sCLU expression was associated with the poor differentiation, TNM stage, and considered as an independent prognostic factor for HCC patients. Furthermore, silencing sCLU gene transcription inhibited the colony formation and proliferation of HCC cells, with decreasing phosphorylation level of AKT and GSK-3ß in HCCLM3 cells in vitro and significantly suppressed the HCC xenograft growth in vivo, suggesting that sCLU with oncogenicity should be not only an early indicator but also novel potential molecular-targeted therapy for HCC.
RESUMO
Hepatitis B virus (HBV) X protein (HBx) plays a key role in the initiation and progression of HBV infectioninduced hepatocellular carcinoma (HCC). Oncogenic microRNA-21 (miR-21) can be modulated by HBx protein in HCC. However, critical regulator genes in the pathway of HBx-induced miR-21 in HCC remain unclear. This study aimed to investigate the role of HBx-induced miR-21 in the apoptosis of HCC cells. In the study, interleukin-12 (IL-12) was demonstrated as a direct target of miR-21 by dualluciferase report assay, and miR-21 was highly expressed in HCC cells (HepG2 and HepG2 2.2.15) compared to L02 cells, but IL-12 was weakly expressed as detected by real-time quantitative PCR (RT-qPCR). Furthermore, miR-21 mimics, inhibitor, HBx-targeted siRNA, and the HBx overexpression vector (pHBx) were used to observe the regulatory effects of HBx-induced miR-21 via IL-12, and cell apoptosis was assessed. The results showed that overexpression of HBx resulted in the inhibition of IL-12. A high level of miR-21 resulted in a significant increase in proliferation and a decrease in IL-12 expression. Inhibition of miR-21 resulted in a significant increase in apoptosis and increased IL-12 expression. The results suggest that HCC cell apoptosis was suppressed at least partially through HBx-induced miR-21 by targeting IL-12.
Assuntos
Carcinoma Hepatocelular/genética , Interleucina-12/genética , Neoplasias Hepáticas/genética , MicroRNAs/genética , Transativadores/genética , Apoptose/genética , Carcinoma Hepatocelular/complicações , Carcinoma Hepatocelular/terapia , Carcinoma Hepatocelular/virologia , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica , Vetores Genéticos/uso terapêutico , Células Hep G2 , Hepatite B/complicações , Hepatite B/genética , Hepatite B/virologia , Vírus da Hepatite B/genética , Vírus da Hepatite B/patogenicidade , Humanos , Interleucina-12/biossíntese , Neoplasias Hepáticas/complicações , Neoplasias Hepáticas/virologia , MicroRNAs/biossíntese , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/uso terapêutico , Transativadores/antagonistas & inibidores , Transativadores/uso terapêutico , Proteínas Virais Reguladoras e AcessóriasRESUMO
The upregulation of secretory clusterin (sCLU) is associated with tumor progression by contributing to angiogenesis, chemo-resistance, cell survival, and metastasis. However, its diagnostic or prognostic values for hepatocellular carcinoma (HCC) still remain to be clarified. The average serum sCLU level analyzed by an enzyme-linked immunosorbent assay was significantly higher (P < 0.001) in HCC patients than that in any of cases with cirrhosis, chronic hepatitis, or healthy control. The area under receiver operating characteristic curve and diagnostic sensitivity were 0.75 and 74.7 % in sCLU, and 0.74 and 58.7 % in α-fetoprotein (AFP), respectively. The combining detections of sCLU and AFP rose up to 90.7 % for HCC diagnosis. In liver, sCLU by immunohistochemistry was significantly higher (P < 0.001) in the HCC (77.3 %) group than that in their para-cancerous group (33.3 %). Abnormal serum or tissue sCLU expression was closely associated with tumor-node-metastasis (TNM) classification of malignant tumors and lymph node metastasis, as an independent prognosis factor (hazard ratio, 2.287; 95 % confidence interval, 1.044-5.007; P = 0.039), and higher sCLU expression significantly correlated (χ (2) = 4.252, P = 0.039) with poor survival of HCC patients analyzed by multivariate Cox regression or Kaplan-Meier method, suggesting that abnormal sCLU expression associated with tumor progression could be a potential diagnostic and prognostic biomarker for HCC.
Assuntos
Biomarcadores Tumorais , Carcinoma Hepatocelular/sangue , Carcinoma Hepatocelular/diagnóstico , Clusterina/sangue , Neoplasias Hepáticas/sangue , Neoplasias Hepáticas/diagnóstico , Adulto , Idoso , Biomarcadores , Carcinoma Hepatocelular/mortalidade , Feminino , Humanos , Estimativa de Kaplan-Meier , Hepatopatias/sangue , Neoplasias Hepáticas/mortalidade , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica , Estadiamento de Neoplasias , Curva ROC , Reprodutibilidade dos Testes , Carga TumoralRESUMO
AIM: To investigate small interfering RNA (siRNA)-mediated inhibition of nuclear factor-kappa B (NF-κB) activation and multidrug-resistant (MDR) phenotype formation in human HepG2 cells. METHODS: Total RNA was extracted from human HepG2 or LO2 cells. NF-κB/p65 mRNA was amplified by nested reverse transcription polymerase chain reaction and confirmed by sequencing. NF-κB/p65 was analyzed by immunohistochemistry. Specific-siRNA was transfected to HepG2 cells to knock down NF-κB/p65 expression. The effects on cell proliferation, survival, and apoptosis were assessed, and the level of NF-κB/p65 or P-glycoprotein (P-gp) was quantitatively analyzed by enzyme-linked immunosorbent assay. RESULTS: HepG2 cells express NF-κB/p65 and express relatively less phosphorylated p65 (P-p65) and little P-gp. After treatment of HepG2 cells with different doses of doxorubicin, the expression of NF-κB/p65, P-p65, and especially P-gp were dose-dependently upregulated. After HepG2 cells were transfected with NF-κB/p65 siRNA (100 nmol/L), the expression of NF-κB/p65, P-p65, and P-gp were downregulated significantly and dose-dependently. The viability of HepG2 cells was decreased to 23% in the combination NF-κB/p65 siRNA (100 nmol/L) and doxorubicin (0.5 µmol/L) group and 47% in the doxorubicin (0.5 µmol/L) group (t = 7.043, P < 0.001). CONCLUSION: Knockdown of NF-κB/p65 with siRNA is an effective strategy for inhibiting HepG2 cell growth by downregulating P-gp expression associated chemosensitization and apoptosis induction.
Assuntos
Antibióticos Antineoplásicos/farmacologia , Carcinoma Hepatocelular/terapia , Doxorrubicina/farmacologia , Resistência a Múltiplos Medicamentos/genética , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias Hepáticas/terapia , Interferência de RNA , RNA Interferente Pequeno/genética , Terapêutica com RNAi , Fator de Transcrição RelA/genética , Transcrição Gênica , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Apoptose/efeitos dos fármacos , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Regulação Neoplásica da Expressão Gênica , Células Hep G2 , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Masculino , RNA Interferente Pequeno/metabolismo , Fatores de Tempo , Fator de Transcrição RelA/metabolismo , TransfecçãoRESUMO
Hepatic annexin A2 (ANXA2) orchestrates multiple biologic processes and clinical symptoms and plays a key role in development, metastasis, and drug resistance of lethal hepatocellular carcinoma (HCC). However, the prognostic significance of ANXA2 for HCC has not been elucidated up to now. In this study, ANXA2 was frequently found to be up-regulated in HCC tissues compared with benign liver disease (BLD) tissues, which was consistent with the results in serum samples and tissue specimens of patients with HCC. Furthermore, ANXA2 expression was significantly correlated with differentiated degree, intrahepatic metastasis, portal vein thrombus, and tumor node metastasis (TNM) staging. More importantly, increased ANXA2 level was first confirmed to be closely associated with shortened overall survival of HCC (χ (2) = 12.872, P = 0.005) and identified as an independent prognostic factor (hazard ratio 1.338, 95 % confidence interval (CI) 1.013 ~ 1.766, P = 0.040), suggesting that ANXA2 up-regulation might represent an acquired metastasis phenotype of HCC, help to screen out high-risk population for HCC, or more effectively treat a subset of postsurgical HCC patients positive for ANXA2.
Assuntos
Anexina A2/sangue , Biomarcadores Tumorais/sangue , Carcinoma Hepatocelular/sangue , Neoplasias Hepáticas/sangue , Adulto , Idoso , Idoso de 80 Anos ou mais , Anexina A2/genética , Biomarcadores Tumorais/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Estimativa de Kaplan-Meier , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Neoplasias/sangue , Neoplasias/genética , Prognóstico , Ativação TranscricionalRESUMO
Abnormal clusterin (CLU) expression is associated with multidrug resistance (MDR) of hepatocellular carcinoma (HCC). In the present study, the CLU expression was analyzed in human hepatoma cells and chemoresistant counterpart HepG2/ADM cells. Compared with L02 cells, the overexpression of cellular CLU was identified in HepG2, HepG2/ADM, SMMC7721, Hep3B ,and PLC cells and relatively lower expression in Bel-7404, SNU-739, and MHCC97H cells. Specific short hairpin RNAs (shRNAs) to silence CLU gene transcription were designed, and the most effective sequences were screened. After the HepG2/ADM cells transfected with shRNA-1, the inhibition of CLU expression was 73.68 % at messenger RNA (mRNA) level by real-time quantitative RT-PCR with obvious enhancement in cell chemosensitivity, increasing apoptosis induced by doxorubicin using fluorescence kit, and Rh-123 retention qualified with flow cytometry. Knockdown CLU also significantly decreased the drug efflux pump activity through the depression of MDR1/P-glycoprotein (q = 11.739, P < 0.001). Moreover, silencing CLU led to downregulation of ß-catenin (q = 13.544, P = 0.001), suggesting that downregulation of CLU might be a key point to reverse multidrug resistance of HepG2/ADM cells.
Assuntos
Carcinoma Hepatocelular/genética , Clusterina/genética , Neoplasias Hepáticas/genética , Transcrição Gênica , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/patologia , Clusterina/antagonistas & inibidores , Resistência a Múltiplos Medicamentos/genética , Resistencia a Medicamentos Antineoplásicos/genética , Inativação Gênica , Células Hep G2 , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/patologiaRESUMO
OBJECTIVE: To investigate the relationship between and underlying mechanistic pathway of clusterin (CLU) and chemo-resistance ofhepatocellular carcinoma (HCC) cells. METHODS: CLU protein expression in HCC cell lines (Hep3B, SMMC7721, PLC, and HepG2) and HepG2/ADM cells was quantified by western blotting. Four short-hairpin (sh)RNAs designed to block CLU-mRNA were generated, screened by RT-PCR, and transfected into the cells to determine effects of CLU on cell viability and apoptosis. Effects of CLU blockade on drug efflux pump activity were measured by flow cytometry. RESULTS: CLU was found to be over-expressed in HCC cell lines and HepG2/ADM cells. The four shRNAs inhibited CLU-mRNA as follows (vs. levels in untransfected cells): shRNA-1: 73.68% (q =23.011, P < 0.01), shRNA-2: 39.26% (q =11.991, P < 0.01), shRNA-3: 62.36% (q =19.392, P < 0.01), and shRNA-4: 55.35% (q =17.149, P < 0.01). shRNA-mediated depletion of CLU led to increased sensitivity to anti-cancer drugs and increased doxorubicin-induced apoptosis in HepG2/ADM cells, as evidenced by the apoptosis ratio of the shRNA-1 group of 39.28% vs. the apoptosis ratio of the untransfected control group of 4.92%. Silencing of CLU also decreased drug etflux pump activity, and the level of MDR1/P-gp expression was significantly reduced (shRNA-1 group vs.untransfected control group: q =14.604, P < 0.01). CONCLUSION: CLU repression may enhance sensitivity of HCC cells to anti-cancers drugs and represents a potential molecular-target for reversal of multidrug-resistant HCC.
Assuntos
Antineoplásicos/farmacologia , Carcinoma Hepatocelular/metabolismo , Clusterina/metabolismo , Resistencia a Medicamentos Antineoplásicos , Neoplasias Hepáticas/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Apoptose , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Sobrevivência Celular , Clusterina/genética , Regulação para Baixo , Doxorrubicina , Humanos , Neoplasias Hepáticas/patologia , RNA Interferente Pequeno/genética , TransfecçãoRESUMO
BACKGROUND: The up-regulation of hepatic Golgi protein 73 (GP73) is associated with the progression of hepatocellular carcinoma (HCC). However, the exact mechanism and clinical values of its diagnosis and prognosis still need to be clarified. OBJECTIVES: To investigate the clinical values of abnormal liver or circulating GP73 expression and their effect on HCC diagnosis and prognosis. MATERIALS AND METHODS: The expression of GP73 was investigated in 88 cancerous and self-control non-cancerous tissues using tissue microarrays with immunohisto- chemistry and was confirmed by Western blotting. Circulating GP73 levels were detected in the sera of 281 patients with liver diseases using enzyme-linked immunosorbent assay. RESULTS: The levels of circulating GP73 expression in the HCC group were higher than those in any group of benign liver diseases or controls. No significant difference was found between GP73 expression and patients' sex or age, tumor size, or AFP level except for those with hepatitis B virus (HBV) infection or distal metastasis (P < 0.05). The area under the receiver operating characteristic curve, sensitivity, and specificity for HCC diagnosis were 0.881, 78.34%, and 77.59% for GP73 levels over 70 µg/L or 0.754, 71.97%, and 84.48% for alpha-fetoprotein levels over 50 µg/L, respectively. The total incidence of GP73 plus alpha-fetoprotein was up to 87.26% for HCC. A positive GP73 result with brown particles was mainly located in the cytosol, with a few in the nucleus and none in the cell membrane, with abnormal expression in HCC tissues (480.7 ± 148.7) that was significantly higher (t = 10.730, P < 0.001) than those in their non-cancerous tissues (208.0 ± 66.1). The high GP73 expression in HCC was related to lymph node metastasis (χ(2) = 6.940, P = 0.008), gross classification (χ(2) = 6.311, P = 0.012), HBV (χ(2) = 4.803, P = 0.028), tumor node metastasis staging (χ(2) = 4.887, P = 0.027), and five-year survival (χ(2) = 5.206, P = 0.023). CONCLUSIONS: Abnormality of hepatic or circulating GP73 expression should be regarded as an emerging biomarker for HCC diagnosis and prognosis.
RESUMO
BACKGROUND: The prognosis of hepatocellular carcinoma (HCC) is poor and its early diagnosis is of the utmost importance. This study aimed to investigate the values of glypican-3 (GPC-3) expression in the liver and sera and its gene transcription for diagnosis and monitoring of metastasis of HCC. METHODS: Liver GPC-3 was analyzed in HCC tissues from 36 patients by immunohistochemistry and Western blotting. GPC-3 mRNA from circulating peripheral blood mononuclear cells from 123 HCC patients or 246 patients with other diseases or 36 HCC tissues was amplified by RT-PCR, quantitative real-time PCR, and confirmed by DNA sequencing. Circulating GPC-3 level was detected by ELISA. RESULTS: The increasing expression of GPC-3 was observed from non-cancerous to cancerous tissues, with brown granule-like staining localized in tumor parts of atypical hyperplasia and HCC formation. The positive rate of GPC-3 was 80.6% in HCC, 41.7% in their paracancerous tissues, and none in distal cancerous tissues (P<0.001), with no significant difference in differentiation grade and tumor number except for size (Z=2.941, P=0.003). Serum GPC-3 was detected only in HCC (52.8%) and significant difference was found between GPC-3 and tumor size (X2=6.318, P=0.012) or HBV infection (X2=23.362, P<0.001). Circulating GPC-3 mRNA was detected in 70.7% of HCC tissues, with relation to TNM stage, periportal cancerous embolus, and extra-hepatic metastasis (P<0.001). The combination of circulating GPC-3, GPC-3 mRNA and alpha-fetoprotein is of complementary value for HCC diagnosis (94.3%). CONCLUSION: Both GPC-3 overexpression and GPC-3 mRNA abnormality could be used as markers for the diagnosis of HCC and monitoring its metastasis.
Assuntos
Carcinoma Hepatocelular/sangue , Carcinoma Hepatocelular/genética , Glipicanas/genética , Neoplasias Hepáticas/sangue , Neoplasias Hepáticas/genética , RNA Mensageiro/sangue , alfa-Fetoproteínas/análise , Adulto , Idoso , Idoso de 80 Anos ou mais , Western Blotting , Carcinoma Hepatocelular/secundário , Estudos de Casos e Controles , Distribuição de Qui-Quadrado , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Imuno-Histoquímica , Neoplasias Hepáticas/química , Neoplasias Hepáticas/patologia , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Prognóstico , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de DNA , Regulação para CimaRESUMO
Hepatocellular carcinoma (HCC) is a highly chemoresistant cancer with no effective systemic therapy. Despite of surgical or locoregional therapies, prognosis remains poor because of high tumor recurrence or progression, and currently, there are no well-established effective adjuvant therapies. Glypican-3 (GPC-3) is specifically overexpressed in hepatoma and perhaps is a valuable molecular target for HCC therapy. In this present study, the effect of silencing GPC-3 gene transcription on human HepG2 cell proliferation was investigated by constructing GPC-3 short hairpin RNA (shRNA) plasmid. After HepG2 cells were transfected with the most efficient shRNA, GPC-3 mRNA expression (90.4 %) was inhibited significantly and estimated by fluorescence quantitative reverse transcriptase-polymerase chain reaction, and the result was accordance with downregulation at the protein level. The percentage of the cell proliferation was down to 28.9 % in the shRNA group and 19.9 % in the shRNA plus sorafenib group. The cell cycles were arrested in the G1 phase (65.6 %) and the apoptosis rate was increasing (66.75 %) in the shRNA1 group with significant alteration compared with that in the negative-shRNA group. Specific shRNA might intervene effectively GPC-3 activation and inhibit tumor cell proliferation, suggesting that GPC-3 gene should be a potential molecular target for HCC therapy.
Assuntos
Carcinoma Hepatocelular/patologia , Proliferação de Células , Glipicanas/genética , Neoplasias Hepáticas/patologia , RNA Interferente Pequeno/genética , Transcrição Gênica , Apoptose , Western Blotting , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Ciclo Celular , Glipicanas/antagonistas & inibidores , Glipicanas/metabolismo , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais CultivadasRESUMO
Abnormal expression of insulin-like growth factor II (IGF-II) is associated with the hepatocyte malignant transformation and hepatocellular carcinoma (HCC) progress. In this study, specific IGF-II miRNA plasmids were constructed and transfected to HepG2 cells to knockdown IGF-II expression for observing effects on the cell proliferation, survival, apoptosis, angiogenesis, and anchorage-independent colony formation. IGF-II mRNA was evaluated by quantitative real-time polymerase chain reaction, and the level of IGF-II or vascular endothelial growth factor (VEGF) was quantitatively analyzed by ELISA. Our data shown that downregulation of IGF-II expression resulted in the viability alteration, proliferation inhibition, and apoptosis occurrence of HepG2 cells. The level of VEGF expression in the supernatant of HepG2 cells in the IGF-II-miRNA-transfected group was significantly decreasing (P < 0.01) than those in the untransfected group or the miRNA-neg-transfected group, with the susceptibility to anoikis and decreasing of anchorage-independent colony formation of HepG2 cells. Thus, we conclude that IGF-II is a potential molecular target for HCC gene therapy.
Assuntos
Comunicação Autócrina/genética , Fator de Crescimento Insulin-Like II/genética , Neovascularização Patológica/genética , Apoptose/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Proliferação de Células , Sobrevivência Celular/genética , Regulação Neoplásica da Expressão Gênica , Células Hep G2 , Humanos , Fator de Crescimento Insulin-Like II/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neovascularização Patológica/metabolismo , Interferência de RNA , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
BACKGROUND: Hepatocellular carcinoma (HCC) is characterized by a multi-cause, multi-stage and multi-focus process of tumor progression. Its prognosis is poor and early diagnosis is of utmost importance. This study was undertaken to investigate the dynamic expression of oncofetal antigen glypican-3 (GPC-3) and GPC-3 mRNA in hepatocarcinogenesis and to explore their early diagnostic value for HCC. METHODS: A hepatoma model was induced in male Sprague-Dawley rats with 0.05% 2-fluorenylacetamide and confirmed by hematoxylin and eosin staining and gamma-glutamyltransferase (GGT) expression. Total RNA was purified and transcribed into cDNA by reverse transcription. Fragments of the GPC-3 gene were amplified by nested RT-PCR, and confirmed by sequencing. GPC-3 was analyzed by immunohistochemistry, Western blotting or ELISA. RESULTS: Positive GPC-3 expression showed as brown granule-like staining localized in the cytoplasm. Histological examination of hepatocytes revealed three morphological stages of granule-like degeneration, atypical hyperplasia (precancerous), and cancer formation, with a progressive increase of liver total RNA and GGT expression. The incidence of liver GPC-3 mRNA and GPC-3, and serum GPC-3 was 100%, 100% and 77.8% in the HCC group, 100%, 100%, and 66.7% in the precancerous group, 83.3%, 83.3%, and 38.9% in the degeneration group, and no expression in the liver or blood of the control group, respectively. There was a positive correlation between liver GPC-3 mRNA and total RNA level (r=0.475, P<0.05) or liver GPC-3 (r=1.0, P<0.001) or serum GPC-3 (r=0.994, P<0.001). CONCLUSION: Abnormal oncofetal antigen GPC-3 and GPC-3 mRNA expression in hepatocarcinogenesis may be promising molecular markers for early diagnosis of HCC.