[show abstract][hide abstract] ABSTRACT: BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most aggressive malignancies in humans, and its prognosis is generally poor even after surgery. Many advances have been made to understand the pathogenesis of HCC; however, the molecular mechanisms that lead to hepatocarcinogenesis and progression are still not clearly understood. METHODS: The expression of DACT2 in specimens from 30 paired HCCs and an additional 61 HCC patients after liver transplantation was evaluated by quantitative RT-PCR and immunohistochemical analysis. We investigated the methylation status of the DACT2 promoter region. We also analyzed the alterations of the cell cycle, migration and invasion after DACT2 knockdown. RESULTS: The expression level of DACT2 was significantly lower in HCC tissues than in non-cancerous tissues. Reduced DACT2 expression was associated with large tumor size. DACT2 transcripts were at low levels in hypermethylated liver cancer cells and were restored by exposure to a demethylating agent. Reduced expression of DACT2 in MHCC97L cells induced G1/S arrest, increased cell proliferation, and promoted cell invasion. CONCLUSIONS: Our study suggests that DACT2 is silenced by promoter hypermethylation, and reduced DACT2 can promote liver cancer progression. DACT2 may serve as a novel tumor suppressor gene in HCC.
World Journal of Surgical Oncology 03/2013; 11(1):57. · 1.09 Impact Factor
[show abstract][hide abstract] ABSTRACT: Metastasis-associated lung adenocarcinoma transcript 1(MALAT1), a long non-coding RNA (lncRNA), is up-regulated in many solid tumors and associated with cancer metastasis and recurrence. However, its role in hepatocellular carcinoma (HCC) remains poorly understood. In the present study, we evaluated the expression of MALAT1 by quantitative real-time PCR in 9 liver cancer cell lines and 112 HCC cases including 60 cases who received liver transplantation (LT) with complete follow-up data. Moreover, small interfering RNA (siRNA) was used to inhibit MALAT1 expression to investigate its biological role in tumor progression. We found that MALAT1 was up-regulated in both cell lines and clinical tissue samples. Patients with high expression level of MALAT1 had a significantly increased risk of tumor recurrence after LT, particularly in patients who exceeded the Milan criteria. On multivariate analysis, MALAT1 was an independent prognostic factor for predicting HCC recurrence (hazard ratio, 3.280, P = 0.003).In addition, inhibition of MALAT1 in HepG2 cells could effectively reduce cell viability, motility, invasiveness, and increase the sensitivity to apoptosis. Our data suggest that lncRNA MALAT1 play an important role in tumor progression and could be a novel biomarker for predicting tumor recurrence after LT and serve as a promising therapeutic target.
Medical Oncology 06/2011; 29(3):1810-6. · 2.14 Impact Factor
[show abstract][hide abstract] ABSTRACT: O-linked-ß-N-acetylglucosamine (O-GlcNAc) modification is a crucial post-translational modification. The enzymes responsible for the addition and removal of O-GlcNAc have been identified as O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). In this study, O-GlcNAcylation level was examined in forty hepatocellular carcinoma (HCC) tissues of patients who underwent liver transplantation (LT) and ten healthy liver tissues by immunohistochemistry analysis. We also examined the expression of OGT and OGA in sixty HCC samples using real-time reverse-transcription polymerase chain reaction and analyzed their correlations with clinical parameters and prognosis in sixty HCC patients treated with LT. Additionally, the global O-GlcNAcylation level was altered through OGT and OGA silencing in the HCC cell line, and the effects of O-GlcNAcylation on cancer malignancy were investigated. We found that the global O-GlcNAcylation levels were significantly elevated in HCC tissues than that in healthy liver tissues (P = 0.031); moreover, O-GlcNAcylation was significantly enhanced in the tumor tissues of patients who had suffered from HCC recurrence after LT compared with those who had not (P = 0.046). Importantly, low expression of OGA was an independent prognostic factor for predicting tumor recurrence of HCC following LT (P = 0.041, hazard ratio, 0.438), especially in AFP low patients. In vitro assays demonstrated that O-GlcNAcylation play important roles in migration, invasion, and viability of HCC cells, partly through regulating E-cadherin, MMP1, MMP2, and MMP3 expression. Altogether, these results suggest that O-GlcNAcylation might play important roles in HCC formation and progression and may be a potential marker to predict patient risk of recurrence after LT and a valuable target for therapy.
Medical Oncology 04/2011; 29(2):985-93. · 2.14 Impact Factor
[show abstract][hide abstract] ABSTRACT: Mesenchymal stem cells (MSCs) and fibroblasts have intimate relationships, and the phenotypic homology between fibroblasts and MSCs has been recently described. The aim of this study was to investigate the hepatic differentiating potential of human fibroblasts in cirrhotic liver.
The phenotypes of fibroblasts in cirrhotic liver were labeled by biological methods. After that, the differentiation potential of these fibroblasts in vitro was characterized in terms of liver-specific gene and protein expression. Finally, an animal model of hepatocyte regeneration in severe combined immunodeficient (SCID) mice was created by retrorsine injection and partial hepatectomy, and the expression of human hepatocyte proteins in SCID mouse livers was checked by immunohistochemical analysis after fibroblast administration.
Surface immunophenotyping revealed that a minority of fibroblasts expressed markers of MSCs and hepatic epithelial cytokeratins as well as alpha-smooth muscle actin, but homogeneously expressed vimentin, desmin, prolyl 4-hydroxylase and fibronectin. These fibroblasts presented the characteristics of hepatocytes in vitro and differentiated directly into functional hepatocytes in the liver of hepatectomized SCID mice.
This study demonstrated that fibroblasts in cirrhotic liver have the potential to differentiate into hepatocyte-like cells in vitro and in vivo. Our findings infer that hepatic differentiation of fibroblasts may serve as a new target for reversion of liver fibrosis and a cell source for tissue engineering.
[show abstract][hide abstract] ABSTRACT: The long noncoding RNA HOTAIR has been reported as a poor prognostic biomarker in patients with breast cancer. The aim of the present study is to examine the expression pattern of HOTAIR in hepatocellular carcinoma (HCC) and its clinical significance as well as its biological role in tumor progression.
We examined the expression of HOTAIR in 110 HCC samples using real-time reverse transcription-polymerase chain reaction and analyzed its correlation with clinical parameters and prognosis in 60 HCC patients that have undergone liver transplantation (LT). Suppression of HOTAIR using siRNA was performed to explore its roles in tumor progression.
The expression level of HOTAIR in cancer tissues was higher than in adjacent noncancerous tissues. High expression level of HOTAIR was an independent prognostic factor for predicting HCC recurrence in LT patients (P = .001, hazard ratio, 3.564). Furthermore, in patients exceeding the Milan criteria, those with a high expression level of HOTAIR revealed a significantly shorter recurrence-free survival. Moreover, siRNA suppression of HOTAIR in a liver cancer cell line reduced cell viability and cell invasion, sensitized TNF-α induced apoptosis, and increased the chemotherapeutic sensitivity of cancer cells to cisplatin and doxorubicin.
The high expression level of HOTAIR in HCC could be a candidate biomarker for predicting tumor recurrence in HCC patients who have undergone liver transplant therapy and might be a potential therapeutic target.
Annals of Surgical Oncology 02/2011; 18(5):1243-50. · 4.12 Impact Factor
[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.
[show abstract][hide abstract] ABSTRACT: Histone deacetylases (HDACs) have been reported to be poor prognostic indicators in patients with cancer. However, no data are available for the role of single nucleotide polymorphism (SNP) of class I HDAC in hepato-cellular carcinoma (HCC). Therefore, we investigated the association of class I HDAC isoforms genomic polymorphisms with risk of HCC and tumor recurrence following liver transplantation (LT).
One hundred and ninety-six Chinese subjects consisting of 97 HCC patients and 99 controls were enrolled in this study. Nine polymorphisms of the HDAC1, HDAC2, and HDAC3 gene (rs2530223, rs1741981, rs2547547, rs13204445, rs6568819, rs10499080, rs11741808, rs2475631, rs11391) were examined using Applied Biosystems SNaP-Shot and TaqMan technology.
We found no significant difference in genotype frequencies between the HCC cases and controls. In terms of tumor recurrence following LT, patients carrying the T allele of HDAC1 SNP rs1741981 showed a favorable outcome for recurrence free survival when compared with patients homozygous for CC. In addition, the same significant trend was observed in HDAC3 SNP rs2547547. Kaplan-Meier analysis showed that the combination of the T variant allele (CT+TT) of HDAC1 SNP rs1741981 and the homozygous TT variant allele of HDAC3 SNP rs2547547 was the most favorable prognostic factor. The risk for postoperative tumor recurrence was about 2.2-fold lower for patients with this genotype combination compared with carriers of the HDAC1 SNP rs1741981 CC and HDAC3 SNP rs2547547 CT genotype combination (hazard ratio: 2.235, p=0.003).
Our data suggest that combined analysis of HDAC1 SNP rs1741981 and HDAC3 SNP rs2547547 may be a potential genetic marker for HCC recurrence in LT patients.
Clinical Chemistry and Laboratory Medicine 12/2010; 48(12):1785-91. · 3.01 Impact Factor
[show abstract][hide abstract] ABSTRACT: CpG island methylator phenotype (CIMP), in which multiple genes concordantly methylated, has been demonstrated to be associated with progression, recurrence, as well as overall survival in some types of cancer.
We examined the promoter methylation status of seven genes including P16, CDH1, GSTP1, DAPK, XAF1, SOCS1 and SYK in 65 cases of HCC treated with LT by methylation-specific PCR. CIMP+ was defined as having three or more genes that are concordantly methylated. The relationship between CIMP status and clinicopathological parameters, as well as tumor recurrence was further analyzed.
CIMP+ was more frequent in HCC with AFP > 400 ng/ml than those with AFP <or= 400 ng/ml (P = 0.017). In addition, patients with CIMP+ were prone to have multiple tumor numbers than those with CIMP- (P = 0.007). Patients with CIMP+ tumors had significantly worse recurrence-free survival (RFS) than patients with CIMP-tumors by Kaplan-Meier estimates (P = 0.004). Multivariate analysis also revealed that CIMP status might be a novel independent prognostic factor of RFS for HCC patients treated with LT (HR: 3.581; 95% CI: 1.473-8.710, P = 0.005).
Our results suggested that CIMP could serve as a new prognostic biomarker to predict the risk of tumor recurrence in HCC after transplantation.
[show abstract][hide abstract] ABSTRACT: Recent studies have shown that high expression levels of class I histone deacetylases (HDACs) correlate with malignant phenotype and poor prognosis in some human tumors. However, the expression patterns and prognostic role of class I HDAC isoforms in hepatocellular carcinoma (HCC) remain unclear.
The expression patterns and clinical significance of class I HDAC isoforms were assessed by immunohistochemistry in a cohort of 43 hepatitis B virus-associated HCC patients treated with liver transplantation. In addition, the effects of HDAC inhibition on HCC cell behavior were investigated by knockdown of the HDAC isoform with short interfering RNA. Class I HDACs were highly expressed in a subset of HCCs with positivity for HDAC1 in 51.2%, HDAC2 in 48.8%, and HDAC3 in 32.6% of cases. The expression levels of HDAC isoforms were significantly associated with the proliferation index of HCC. Kaplan-Meier curves showed that a high expression level of HDAC2 or HDAC3 implicated significantly reduced recurrence-free survival. Cox proportional hazards model analysis revealed HDAC3 overexpression was an unfavorable independent prognostic factor (P = 0.002; HR 3.907). In vitro, inhibition of HDAC2 and HDAC3, but not HDAC1, suppressed proliferation and the invasiveness of liver cancer cells.
Our findings demonstrate that HDAC3 plays a significant role in regulating tumor cell proliferation and invasion, and it could be served as a candidate biomarker for predicting the recurrence of hepatitis B virus-associated HCC following liver transplantation and a potential therapeutic target.
PLoS ONE 01/2010; 5(12):e14460. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Vascular endothelial growth factor (VEGF), an important regulator of angiogenesis and vascular permeability, is involved in various steps of many malignancies. Gene polymorphisms within the gene encoding VEGF have been shown to be independently associated with an adverse outcome in various malignancies including hepatocellular carcinoma (HCC) with resection. However, no data are available for HCC treated with liver transplantation (LT). Therefore, we investigated association of VEGF genomic polymorphisms with risk for developing HCC and tumor recurrence after LT.
Seven polymorphisms in the VEGF gene (rs699947, rs1570360, rs2010963, rs3024997, rs3025010, rs3025035, rs3025039) were examined in 93 HCC patients treated with LT and 99 controls using Applied Biosystems SNaP-Shot and TaqMan technology. Cox proportional hazard model was used to estimate the hazard ratios associated with polymorphisms.
The association between rs3025035 and recurrence was significant (p=0.003). However, no other SNP in VEGF was associated with recurrence. Interestingly, we found that patients with rs3025035 CT heterozygous was independently associated with a shortened recurrence-free survival (odds ratio: 3.3; 95% confidence interval: 1.8-6.0; p<0.001).
Our data suggest that polymorphism rs3025035 in the VEGF gene may be a potential genetic marker for HCC recurrence in LT patients.
Archives of medical research 10/2009; 40(7):565-70. · 1.88 Impact Factor
[show abstract][hide abstract] ABSTRACT: Transcriptional regulation of the putative tumor suppressor gene X-linked inhibitor of apoptosis protein-associated factor 1 (XAF1) by promoter methylation has been related to tumor progression in gastric and bladder cancer. The aim of this study was to investigate the methylation status and expression level of XAF1 in human hepatitis B virus (HBV)-associated hepatocellular carcinoma (HCC) treated with liver transplantation (LT), and to evaluate potential predictive value for tumor recurrence.
The expression level and methylation status of XAF1 in three liver cancer cell lines (SMMC-7721, HepG2, and Hep3B) and 65 cases of HBV-associated HCC following LT were analyzed by RT-PCR (RT, reverse-transcriptase), immunohistochemistry, and methylation-specific polymerase chain reaction (PCR).
XAF1 transcripts were not observed or present at low levels in liver cancer cell lines and were restored by treatment with demethylating agent 5-aza-2'-deoxycytidine (5-Aza-dC). In vivo, methylation status was associated with protein level of XAF1 (P < 0.001) and serum level of alpha-fetoprotein (AFP) (P = 0.009). The expression pattern of XAF1 was associated with portal vein tumor thrombi (PVTT), preoperative AFP level, tumor size, and recurrence. Multivariate analysis revealed that expression level of XAF1 was an independent factor for predicting recurrence-free survival [hazard ratio 0.237, 95% confidence interval (CI) 0.095-0.592, P = 0.002]. However, no significant association was found between methylation status and the risk of tumor recurrence.
Promoter hypermethylation is a critical, but not the sole, mechanism for gene silencing of XAF1 in HCC. Protein level of XAF1 may serve as a potential biomarker for tumor recurrence after LT.
Annals of Surgical Oncology 11/2008; 15(12):3494-502. · 4.12 Impact Factor
[show abstract][hide abstract] ABSTRACT: To investigate the effects of ciclosporin (CsA) and tacrolimus (FK506) on replication of hepatitis B virus (HBV) in vitro.
HBV genome permanently transfected human liver cancer cells of the line HepG2.2.15 were cultured. CsA and FK506 at different concentrations were added into the culture fluid so as to identify the nontoxic concentrations by MTT method. Then the HepG2.2.15 cells were treated by CsA and FK506 at different nontoxic concentrations respectively for 4 days. ELISA was used to detect the HB surface antigen (HBsAg) and HB e antigen (HBeAg) in the supernatant. The relative replication level of HBV DNA was detected by slot blot analysis.
MTT method confirmed that the nontoxic concentrations of CsA and FK506 were 0-40.0 microg/ml and 0-400 ng/ml respectively. After the treatment of CsA at the concentration of 1.3, 2.5, and 5.0 microg/ml, in comparison to the control group, the suppression rates of HBsAg expression in the HepG2.2.15 cells were 16.5% +/- 9.4%, 21.5% +/- 8.9%, and 33.1% +/- 5.3% respectively (all P < 0.05); the suppression rates of HBeAg expression in the HepG2.2.15 cells were 7.8% +/- 2.2%, 11.0% +/- 2.3%, and 20.8% +/- 1.5% respectively (all P < 0.05); and the HBV DNA replication levels were 56 +/- 16, 42 +/- 11, and 40 +/- 10 respectively (P > 0.05, P < 0.05, and P > 0.05). However, FK506 at different nontoxic concentrations showed no significant inhibitory effect on the levels of HBsAg, HBeAg, and HBV DNA.
CsA dose-dependently inhibits the HBV replication in vitro, and FK506 does not exercise similar effects.