[Show abstract][Hide abstract] ABSTRACT: Background:
Carcinoma associated fibroblasts (CAFs), an important component of tumor microenvironment, are capable of enhancing tumor cells invasion and migration through initiation of epithelial-mesenchymal transition (EMT). MRC-5 fibroblasts are one of the CAFs expressing alpha-smooth muscle actin. It is ascertained that medium conditioned by MRC-5 fibroblasts stimulate motility and invasion of breast cancer cells. However, its role in hepatocellular carcinoma (HCC) is less clear. The aim of our study was to investigate the effect of MRC-5-CM on HCC and explore the underlying mechanisms.
Methods and results:
Using a combination of techniques, the role of MRC-5-CM in HCC was evaluated. We determined that MRC-5-CM induced the non-classical EMT in Bel-7402 and MHCC-LM3 cell lines. Initiation of the non-classical EMT was mainly via quintessential redistribution of α-, β- and γ-catenin, P120 catenin, E-cadherin, and N-cadherin, rather than up-regulation of typical EMT-related transcription factors (i.e., Snail, Twist1, ZEB-1 and ZEB2). We also found that MRC-5-CM potentiated both the migration and invasion of Bel-7402 and MHCC-LM3 cells in mesenchymal movement mode through activation of the α6, β3, β4, β7 integrin/FAK pathway and upregulation of MMP2. The flow cytometric analysis showed that MRC-5-CM induced G1 phase arrest in Bel-7402 cells with a concomitant reduction of S phase cells. In contrast, MRC-5-CM induced S phase arrest in MHCC-LM3 cells with a concomitant reduction of cells in the G2/M phase. MRC-5-CM also inhibited apoptosis in Bel-7402 cells while inducing apoptosis in MHCC-LM3 cells.
Collectively, MRC-5-CM promoted HCC cell motility and invasiveness through initiation of the non-classical EMT, including redistribution of α-, β- and γ-catenin, P120 catenin, E-cadherin, and N-cadherin, activation of the integrin/FAK pathway, and upregulation of MMP2. Hence, MRC-5-CM exerted distinct roles in Bel-7402 and MHCC-LM3 cell viability by regulating cyclins, cyclin dependent kinases (CDKs), CDK inhibitors (CKIs), Bcl-2 family proteins and other unknown mechanosensors.
Preview · Article · Jul 2015 · Journal of Translational Medicine
[Show abstract][Hide abstract] ABSTRACT: Objective:
To analyze the risk factors for biliary complications of liver transplantation from donation after cardiac death (DCD).
Clinical data of 109 patients undergoing liver transplantation from DCD in First Affiliated Hospital of Zhejiang University School of Medicine from October 2010 to October 2013 were studied retrospectively. The risk factors of biliary complications following DCD liver transplantation were analyzed.
Twenty-four (22%) patients developed biliary complications after DCD liver transplantation. Univariate analysis showed that biliary complications were associated with warm ischemia time (P<0.001) and length of ICU stay (P=0.013), but not associated with ABO blood types match (P>0.05). Administration of inotropic agents and fatty liver increased the trend of biliary complications. Multivariate analysis demonstrated that warm ischemia time and length of ICU stay were independent risk factors for predicting biliary complications.
Warm ischemia time and days of ICU stay are independent risk factors for predicting biliary complications after DCD liver transplantation.
No preview · Article · Nov 2014 · Zhejiang da xue xue bao. Yi xue ban = Journal of Zhejiang University. Medical sciences
[Show abstract][Hide abstract] ABSTRACT: Objective:
To investigate the risk factors associated with persistent thrombocytopenia after liver transplantation (LT), and to explore effective measures for prevention.
One hundred and twenty-eight adult patients, who received liver transplantation in our hospital between January 2009 and June 2012 and met the inclusive criteria, were enrolled in the study. The clinical data were retrospectively analyzed, including pre-LT spleen volume, main portal vein size, coronary vein size, platelet and white blood cell levels, total bilirubin level and model of end stage liver disease score. The risk factors associated with persistent thrombocytopenia after LT were evaluated by logistic regression analysis. The effect of simultaneous splenic artery coarctation for high risk patients was evaluated with χ2 test.
Logistic regression analysis showed that per-LT spleen volume larger than 500 ml (P = 0.012, OR=2.789, 95%CI: 1.249-6.227) and portal vein size beyond 15 mm (P = 0.017, OR = 3.124, 95%CI: 1.230-7.933) were independent risk factors for persistent thrombocytopenia after LT. The incidence rate of persistent thrombocytopenia after LT in patients with or without simultaneous splenic artery coarctation were 16.7% (1/6) and 66.7% (32/48), respectively(P < 0.05).
Spleen volume larger than 500 ml and portal vein size beyond 15 mm are risk factors for persistent thrombocytopenia after LT. Simultaneous splenic artery coarctation may reduce the occurrence of persistent thrombocytopenia after LT.
No preview · Article · Nov 2014 · Zhejiang da xue xue bao. Yi xue ban = Journal of Zhejiang University. Medical sciences
[Show abstract][Hide abstract] ABSTRACT: FBW7 is a tumor suppressor which regulates a network of proteins with central roles in cell division, cell growth and differentiation. This study aimed to evaluate the role of FBW7 in chemosensitivity and epithelial-mesenchymal transition (EMT) in different hepatocellular carcinoma (HCC) cell lines and to investigate the relevant underlying mechanisms.
Different human HCC cell lines (Hep3B, Huh-7, and SNU-449) were cultured. The cell viability was evaluated by cell counting kit-8, and FBW7 mRNA transcription and protein expression were quantitated by real-time PCR and Western blotting. Expressions of vimentin (mesenchymal biomarker) and E-cadherin (epithelial biomarker) were evaluated by Western blotting and immunocytochemistry. Cell invasion was assayed by Transwell migration, and FBW7 plasmid or siRNA was used to evaluate the effect of FBW7 overexpression or silencing on cell chemosensitivity.
FBW7 expression affected tumor cell chemosensitivity to doxorubicin and tumor cell invasive capacity in different HCC cell lines. FBW7hi (high FBW7 expression) Hep3B and FBW7mi (median FBW7 expression) Huh-7 cells were more sensitive to doxorubicin and lower in invasive capacity than FBW7lo (low FBW7 expression) SNU-449 cells. Silencing of FBW7 in Huh-7 and Hep3B cells induced the resistance to doxorubicin and enhanced cell invasion, whereas overexpression of FBW7 in SNU-449 cells restored the sensitivity to doxorubicin and significantly reduced invasive capacity. Furthermore, doxorubicin induced EMT toward mesenchyme in HCC cells. Downregulation of FBW7 in Huh-7 and Hep3B cells or upregulation of FBW7 in SNU-449 cells altered the direction of EMT.
The level of FBW7 expression impacted the tumor resistance to doxorubicin and the invasion capability of HCC cells. FBW7 therefore may be a potential target for the chemotherapy of HCC through the regulation of EMT.
No preview · Article · Apr 2014 · Hepatobiliary & pancreatic diseases international: HBPD INT
[Show abstract][Hide abstract] ABSTRACT: Deregulation of microRNA‑200a (miR‑200a) has been observed in different types of diseases, including cancers. However, the exact roles of miR‑200a in hepatocellular carcinoma (HCC) are still largely unknown. We aimed to elucidate the prognostic implications of miR‑200a and its biological function in HCC. Quantitative polymerase chain reaction was used to evaluate miR‑200a expression. Western blotting was performed to evaluate the protein level. Gain-of-function studies were performed to evaluate the roles of miR‑200a in HCC. Our results revealed that miR‑200a was frequently downregulated in HCC. In addition, multivariate analysis confirmed that miR‑200a was significantly associated with the overall survival of HCC patients. In vitro assays demonstrated that miR‑200a suppressed the proliferation of HCC cells by induction of G1 phase arrest. Furthermore, CDK6 was identified as a novel functional target of miR‑200a. Our data indicate that miR‑200a functions as a potential tumor suppressor in HCC.
[Show abstract][Hide abstract] ABSTRACT: Increasing evidence indicates that deregulation of microRNAs (miRNAs) is involved in tumorigenesis. Downregulation of microRNA-503 has been observed in various types of diseases, including cancer. However, the biological function of miR-503 in hepatocellular carcinoma (HCC) is still largely unknown. In this study we aimed to elucidate the prognostic implications of miR-503 in HCC and its pathophysiologic role.
Quantitative reverse transcriptase polymerase chain reaction was used to evaluate miR-503 expression in HCC tissues and cell lines. Western blotting was performed to evaluate the expression of the miR-503 target genes. In vivo and in vitro assays were performed to evaluate the function of miR-503 in HCC. Luciferase reporter assay was employed to validate the miR-503 target genes.
miR-503 was frequently downregulated in HCC cell lines and tissues. Low expression levels of miR-503 were associated with enhanced malignant potential such as portal vein tumor thrombi, histologic grade, TNM stage, AFP level and poor prognosis. Multivariate analysis indicated that miR-503 downregulation was significantly associated with worse overall survival of HCC patients. Functional studies showed miR-503 suppressed the proliferation of HCC cells by induction of G1 phase arrest through Rb-E2F signaling pathways, and thus may function as a tumor suppressor. Further investigation characterized two cell cycle-related molecules, cyclin D3 and E2F3, as the direct miR-503 targets.
Our data highlight an important role for miR-503 in cell cycle regulation and in the molecular etiology of HCC, and implicate the potential application of miR-503 in prognosis prediction and miRNA-based HCC therapy.
Preview · Article · Aug 2013 · Journal of Translational Medicine
[Show abstract][Hide abstract] ABSTRACT: It has been reported bone marrow mesenchymal stem cells (BMMSCs) facilitate liver regeneration after toxic injuries. However, the effect of BMMSCs on liver regeneration after massive hepatectomy is barely studied. Here we explored if infusion of BMMSCs promotes liver regeneration in a rat massive hepatectomy model.
Hypoxia preconditioning was achieved by culturing BMMSCs under hypoxia environment. 85% hepatectomy was performed and hypoxia or normoxia preconditioned BMMSCs were infused into the portal vein. A group of rats received vascular endothelial growth factor (VEGF) neutralizing antibody peri-operatively, underwent 85% hepatectomy and subsequently infusion of hypoxia preconditioned BMMSCs to verify the role of VEGF in BMMSC's effects on liver regeneration. Liver samples were collected and liver regeneration was evaluated post-operatively.
Hypoxia preconditioning enhanced the expression of VEGF in BMMSCs in vitro. Infusion of BMMSCs promoted proliferation of hepatocyte as reflected by elevated cyclinD1 expression and proliferating cell nuclear antigen (PCNA)-positive hepatocytes. However, BMMSC infusion didn't improve serum albumin level, liver weight/body weight ratio (LBWR), and survival after operation. Infusion of hypoxia preconditioned BMMSCs significantly elevated cyclinD1, PCNA-positive hepatocytes, LBWR, and survival compared with normoxia preconditioned BMMSCs, accompanied by increased serum albumin level. And the level of VEGF in liver homogenate was much higher in hypoxia preconditioned BMMSC treated animals than other groups. In addition, the peri-operative injection of VEGF neutralizing antibody significantly blocked the therapeutic effects of hypoxia preconditioned BMMSCs on liver injury and regeneration in this model.
Hypoxia preconditioned BMMSCs enhanced liver regeneration after massive hepatectomy in rats, possibly by up-regulating the level of VEGF.
[Show abstract][Hide abstract] ABSTRACT: The epithelial-to-mesenchymal transition (EMT) and mesenchymal-to-epithelial transition (MET) play pivotal roles in metastasis of epithelial cancers. The distinction between them has shed new light on the molecular mechanisms of tumor metastasis. Recently, tumor microenvironment (TM) has been identified as one of the most potent inducers of EMT and MET. TM is characterized by its complexity and flexibility. The purpose of this study was to ascertain the exact effect of each distinct TM component on the evolution hepatocellular carcinoma (HCC) metastasis.
Two different cell culture models were used. The HCC cell line Bel-7402 was co-cultured with the normal liver cell line HL-7702 or with the retinal vascular endothelial cell line RF/6A in double-layer six-well plates, imitating the direct interaction between tumor-host cells and tumor cells. Bel-7402 was also cultured in the conditioned medium (CM) of the human lung fibroblast cell line MRC-5, HL-7702 or RF/6A, imitating an indirect interaction. Integrin beta1, beta3, beta4, beta7, laminin beta3, E-cadherin and Snail levels were measured by quantitative RT-PCR in tumor sepecimens from 42 resected HCC.
We found that Bel-7402 cells co-cultured with HL-7702 or RF/6A cells were induced to undergo MET. The expression of E-cadherin, alpha-catenin and beta-catenin was up-regulated, accompanied with a strengthened E-cadherin/catenin complex on the membrane of co-cultured Bel-7402 cells. Consequently, the invasion and migration ability of cells was declined. Conversely, Bel-7402 cells cultured in conditioned medium from MRC-5 cells underwent an EMT-like transformation as the cells became elongated with increased invasion and migration ability. Furthermore, we demonstrated that HL-7702 cells could generally inhibit the tumorigenicity and viability of Bel-7402 cells. We also found that integrin beta1 expression was negatively associated with capsular formation, and that integrin beta4 expression was negatively associated with CK19 expression.
Our findings highlight the strong influences exerted by TM on tumor progression through EMT and MET by impacting the expression of adhesion molecules, including the E-cadherin/catenin complex, laminins and integrins.
Preview · Article · Jul 2013 · Journal of Translational Medicine
[Show abstract][Hide abstract] ABSTRACT: It has been demonstrated that Ring finger protein 43 (RNF43) is overexpressed in colorectal cancer and mediates cancer cell proliferation; however, its role in hepatocellular carcinoma (HCC) remains unknown. In this study, we found that RNF43 was frequently overexpressed in HCC, and this overexpression was correlated with positive vascular invasion, poor tumor differentiation and advanced tumor stage. Functional studies showed that knockdown of RNF43 could induce apoptosis and inhibit proliferation, invasion, colony formation and xenograft growth of HCC cells. Microarray-based gene profiling demonstrated a total of 229 genes differentially expressed after RNF43 knockdown, many of which are involved in oncogenic processes such as cell proliferation, cell adhesion, cell motility, cell death, DNA repair and so on. These results suggest that RNF43 is involved in tumorigenesis and progression of HCC, and antagonism of RNF43 may be beneficial for HCC treatment.
No preview · Article · Nov 2012 · Molecular Cancer Therapeutics
[Show abstract][Hide abstract] ABSTRACT: Crigler-Najjar syndrome type I (CNS I) is a very rare autosomal recessive inherited disease that liver transplantation can properly deal with.
We present one case of an 18-month-old child with CNS I diagnosed by clinical findings and genetic detecting. LTx was performed 5 days after kernicterus broke out and neurological symptoms were successfully reversed.
Magnetic resonance imaging and magnetic resonance spectroscopy showed encouraging results that brain pathology had a trend to return to normal in 1-year follow-up, combined with electroencephalogram and motor development estimate studies.
Liver transplantation can cure CNS I with reversible neurological symptoms to some extent in time. Magnetic resonance spectroscopy may be a future option of predicting brain conditions and selecting suitable patients with CNS I for transplantation.
No preview · Article · Oct 2012 · Hepatobiliary & pancreatic diseases international: HBPD INT
[Show abstract][Hide abstract] ABSTRACT: The existence of microvascular invasion (MVI) formation is one of the most important risk factors predicting poor outcome in hepatocellular carcinoma (HCC) and its mechanism remains largely unknown. Epithelial-Mesenchymal Transition (EMT) has been suggested to be involved in many steps of the invasion-metastasis cascade. To elucidate the possible contribution of EMT to MVI, we initially evaluated the expression of 8 EMT-related transcription factors (TFs) in HCC patients with or without MVI and found that FOXC1 expression was significantly higher in patients with MVI than those without MVI (P < 0.05). Knockdown of FOXC1 expression in HCC cells resulted in a partial conversion of their EMT progresses, mainly regulating the mesenchymal component. Ectopic expression of snail, twist or TGF-β1 could induce expression of FOXC1, but none of the expression of snail, twist, slug or TGF-β was consistently down-regulated in response to FOXC1 silencing, suggesting FOXC1 might operate the downstream of other EMT regulators. In addition, knockdown of FOXC1 expression led to cytoskeleton modification accompanied by decreased ability of cell proliferation, migration, and invasion. Meanwhile, some matrix metalloproteinases (MMPs) and VEGF-A were also simultaneously down-regulated. Together, our findings demonstrate that FOXC1 is one of candidate predictive markers of MVI, and that inhibition of FOXC1 expression can partially reverse EMT program, offering a potential molecular therapeutic target for reducing tumor metastasis in HCC patients.
Preview · Article · Sep 2012 · International journal of biological sciences
[Show abstract][Hide abstract] ABSTRACT: Cyclin-dependent kinase inhibitor 3 (CDKN3) belongs to the protein phosphatases family and has a dual function in cell cycling. The function of this gene has been studied in several kinds of cancers, but its role in human hepatocellular carcinoma (HCC) remains to be elucidated. In this study, we found that CDKN3 was frequently overexpressed in both HCC cell lines and clinical samples, and this overexpression was correlated with poor tumor differentiation and advanced tumor stage. Functional studies showed that overexpression of CDKN3 could promote cell proliferation by stimulating G1-S transition but has no impact on cell apoptosis and invasion. Microarray-based co-expression analysis identified a total of 61 genes co-expressed with CDKN3, with most of them involved in cell proliferation, and BIRC5 was located at the center of CDKN3 co-expression network. These results suggest that CDKN3 acts as an oncogene in human hepatocellular carcinoma and antagonism of CDKN3 may be of interest for the treatment of HCC.
No preview · Article · Feb 2012 · Biochemical and Biophysical Research Communications
[Show abstract][Hide abstract] ABSTRACT: N-myc downstream-regulated gene 1 (NDRG1) has been reported to be a multifunctional protein associated with carcinogenesis, however, the cellular function of NDRG1 remains elusive in human cancers. Here, our proteomics profile analysis of HCC tissues with different metastatic capabilities revealed that NDRG1 was correlated with metastasis and recurrence in HCC patients after liver transplantation (LT). Immunohistochemical staining of 143 HCC patients after LT showed that NDRG1-positive expression had poor prognosis, either for shorter disease-free survival or overall survival (P < 0.001), compared with NDRG1-negative expression. Multivariate analysis confirmed NDRG1 as an independent prognostic value (P < 0.001). In addition, in vitro experiments HCC cells with small interfering RNA against NDRG1 significantly suppressed its proliferation, colony formation, invasion and migration ability. Microarray analysis revealed that NDRG1 modulated the expression of genes associated with transmembrane transporter activity, chemoattractant activity, immune response, cell adhesion and cell proliferation process. Taken together, these results suggested that NDRG1 was an important molecule in controlling HCC metastasis and thus suggested as a novel biomarker for predicting HCC recurrence after LT.
[Show abstract][Hide abstract] ABSTRACT: According to the control characteristics of brushless DC motor, a control system based on the LPC1700 ARM is designed, together with the corresponding external circuit. This system fully utilizes the high-performance 32-bit calculation of ARM chips and abundant hardware resources. It is compact in structure, reliable in operation and superior in speed regulation performance.
[Show abstract][Hide abstract] ABSTRACT: With the development of high-throughput screening, a variety of genetic alterations has been found in hepatocellular carcinoma (HCC). Although previous studies on HCC methylation profiles have focused on liver tissue, studies using isolated hepatocytes are rare. The heterogeneity of liver composition may impact the genuine methylation status of HCC; therefore, it is important to clarify the methylation profile of hepatocytes to aid in understanding the process of tumorigenesis.
The global methylation profile of single hepatocytes isolated from liver tissue of hepatitis B virus (HBV) related HCC (HBHC) was analyzed using Illumina Infinium Human Methylation27 BeadChips, and combined bisulfite restriction analysis (COBRA) and bisulfite sequencing were used to validate the 20 significant hypermethylated genes identified. In this study, we found many noteworthy differences in the genome-wide methylation profiles of single hepatocytes of HBHC. Unsupervised hierarchical clustering analysis showed that hepatocyte methylation profiles could be classified according to three cell types: hepatocytes of HCC, adjacent hepatocytes and normal hepatocytes. Among the 20 most hypermethylated genes in the hepatocytes of HBHC, 7 novel genes (WNK2, EMILIN2, TLX3, TM6SF1, TRIM58, HIST1H4Fand GRASP) were found to be hypermethylated in HBHC and hypomethylated in paired adjacent liver tissues; these findings have not been reported in previous studies on tissue samples.
The genome-wide methylation profile of purified single hepatocytes of HBHC was aided in understanding the process of tumorigenesis, and a series of novel methylated genes found in this study have the potential to be biomarkers for the diagnosis and prognosis of HBHC.
[Show abstract][Hide abstract] ABSTRACT: BACKGROUND: Tri-iodothyronine (T3) has been shown to be a hepatic mitogen. The aim of this study was to evaluate the effect of T3 on liver regeneration after 50% partial liver transplantation (pLT) in rats. METHODS: Immediately after pLT, a single dose of T3 (4 mg/kg body weight) was administered. Liver/body weight ratio (LBWR), hepatocyte proliferation (Ki-67), biochemical parameters, and changes in cell cycle related proteins were evaluated. RESULTS: T3 promoted liver regeneration as shown by an increased liver/body weight ratio and Ki-67 proliferation index after pLT. On the transcriptional level, T3-treated rats had an increased expression of cyclin D1 and cyclin A as demonstrated by real time RT-PCR and Western blot. CONCLUSIONS: Exogenous administration of T3 significantly improved liver regeneration after pLT, and therefore it may represent a promising strategy to improve the clinical outcome after living donor liver transplantation in the future.
No preview · Article · May 2011 · Journal of Hepato-Biliary-Pancreatic Sciences
[Show abstract][Hide abstract] ABSTRACT: Approximately 20-40% of hepatocellular carcinoma (HCC) patients who undergo liver transplantation (LT) experience HCC recurrence within 5 years of the operation. Current predictors cannot sufficiently differentiate patients at risk for biochemical recurrence. The aim of the present study was to investigate the methylation status and expression levels of cell adhesion molecule 1 (CADM1) in HCC; to elucidate its regulation mechanisms; and finally, to evaluate the potential predictive value for tumor recurrence. Aberrant hypermethylation of CADM1 was frequently found in HCC cell lines with decreased CADM1 mRNA by bisulfite sequencing PCR. Re-expression of CADM1 was induced by treatment with demethylating agents. The promoter region of CADM1 was identified and the basal promoter activity was located in the -226 to -146 region relative to the transcriptional start site (TSS). Site-directed mutagenesis revealed that the consensus Sp1 binding site located in the basal promoter region was important for mediating CADM1 promoter activity. Furthermore, aberrant hypermethylation of CADM1 was detected in 34 of 82 (41.5%) of HCC tissues. The recurrence rate of the patients with CADM1 methylation was higher compared to that without CADM1 methylation (70.6% versus 33.3%; P=0.001). Multivariate analysis revealed that CADM1 methylation status (HR = 2.788; 95% CI, 1.043-5.063; P=0.010) was an independent prognostic factor for disease-free survival (DFS) of HCC patients treated with LT. In conclusion, CADM1 methylation may be used as a potential predictive biomarker for tumor recurrence of HCC after LT.
No preview · Article · Apr 2011 · Oncology Reports
[Show abstract][Hide abstract] ABSTRACT: Fluvastatin, a lipophilic statin, was known to inhibit proliferation and induce apoptosis in many cancer cells. Its potential anticancer was evaluated in three hepatocellular carcinoma (HCC) cell lines (HepG2, SMMC-7721 and MHCC-97H). Cells were treated with fluvastatin in vitro and its effect on cell proliferation, cell cycle, invasion and apoptosis was determined. Mechanism of apoptosis induced by fluvastatin on HCC cell lines was also investigated through western blotting and mitochondrial membrane potential (MMP) analysis. It was observed that fluvastatin inhibited proliferation of HCC cells by inducing apoptosis and G2/M phase arrest in a dose-dependent manner. The results of cell invasion assay revealed that fluvastatin significantly decreased the invasion potency of HCC cells. A mitochondria-operated mechanism for fluvastatin induced apoptosis might be involved and was supported by Western blotting and MMP analysis. After fluvastatin treatment, expression of Bcl-2 and procaspase-9 were downregulated, cytochrome c (cytosolic extract), Bax and cleaved-caspase-3 protein expression were increased. Furthermore, a breakdown of MMP in HCC cells was observed. To conclude, these results have provided a rationale for clinical investigations of fluvastatin in future as a potential anticancer reagent for growth control of HCC.
Preview · Article · Dec 2010 · Indian journal of experimental biology