Decreased PADI4 mRNA Association with Global Hypomethylation in Hepatocellular Carcinoma During HBV Exposure

To gain insight into the role of peptidylarginine deiminase type 4 (PADI4), we determined the relationship between PADI4 mRNA and global hypomethylation during HBV exposure in hepatocarcinogenesis. We analyzed Line-1 methylation by MSP, and CD133 mRNA by real-time PCR in 74 HCC. The HCC cancer lines (7721, Huh7, and Hep-G2) were treated with 5-Aza-CdR and TSA. PADI4 mRNA were lower in HCC tissues (Mean_??Ct = 1.41) than that in Non-Hcc tissues (Mean_??Ct = 3.10). Expression of PADI4 was elevated in only 20 (27 %) of the 74 HCC patients but decreased in 54 (73 %) of the patients. The declined PADI4 mRNA was significantly associated with Line-1 demethylation in HCC patients. PADI4 mRNA were lower in HCC patients with Line-1 ?MI <?0.15 (Mean_???Ct = ?2.66) than those with Line-1 ?MI >= ?0.15 (Mean_???Ct = ?1.02). The results suggested that HCC showing hypomethylation of Line-1 is considered to be silencing PADI4 mRNA. And the lower PADI4 mRNA was also found in HCC patients with HBV >= 10⁵ (copy/ml) than those with HBV < 10⁵ (copy/ml). After treated by 5-Aza-CdR and TSA, we found that PADI4 mRNA induced significantly by TSA in Huh7 and Hep-G2 cells, but not in 7721 cells. Meanwhile, PADI4 mRNA induced by the combination of 5-Aza-CdR and TSA in HCC cells, and it could no effect for exposure to 5-Aza-CdR alone. The results suggested that decreased PADI4 mRNA is associated with global hypomethylation in HCC during HBV exposure.     

Effect of RECK gene polymorphisms on hepatocellular carcinoma susceptibility and clinicopathologic features

Background

The reversion-inducing-cysteine-rich protein with Kazal motifs (RECK) down-regulation has been confirmed in numerous human cancers and is clinically associated with metastasis. This study investigates the potential associations of RECK single-nucleotide polymorphisms (SNPs) with hepatocellular carcinoma (HCC) susceptibility and its clinicopathologic characteristics.

Methodology/Principal Findings

A total of 135 HCC cancer patients and 501 cancer-free controls were analyzed for four RECK SNPs (rs10814325, rs16932912, rs11788747, and rs10972727) using real-time PCR and PCR-RFLP genotyping analysis. After adjusting for other co-variants, the individuals carrying RECK promoter rs10814325 inheriting at least one C allele had a 1.85-fold [95% confidence interval (CI), 1.03–3.36] risk of developing HCC compared to TT wild type carriers. The HCC patients, who carried rs11788747 with at least one G allele, had a higher distant metastasis risk than wild type probands.

Conclusions

RECK gene polymorphisms might be a risk factor increasing HCC susceptibility and distant metastasis in Taiwan.     

The Suppressive Role and Aberrent Promoter Methylation of BTG3 in the Progression of Hepatocellular Carcinoma

Background

BTG3 (B-cell translocation gene 3) has been identified as a tumor suppressor and hypermethylation contributes to its down-regulation in some tumors, but its role in hepatocellular carcinoma (HCC) remain unknown. This study aimed to detect the expression and methylation status of BTG3 in HCC cell lines or tissues, and determine its function in HCC progression.

Methodology

The expression of BTG3 was detected in HCC cell lines and HCC tissue by real-time RT-PCR, Western blot or immunohistochemistry. The promoter methylation status of BTG3 was measured by using methylation-specific PCR in HCC cell lines. A series of assays were performed to evaluate the effect of BTG3 on proliferation, invasion and cell cycle transition in vitro.

Results

BTG3 expression was lower in HCC cell lines than in hepatocyte cell line LO2 (P<0.05). BTG3 was also down-regulated in HCC tissues. Its expression was positively correlated with differentiation and distant metastasis (P<0.05). Patients with lower BTG3 expression had shorter overall survival time (P=0.029). DNA methylation directed repression of BTG3 mRNA expression in HCC cell lines. BTG3 suppressed proliferation, invasion and induces G1/S cycle arrest of HCC cells in vitro.

Conclusion

Down-regulation of BTG3 due to the promoter hypermethylation is closely associated with proliferation, invasion and cell cycle arrest of HCC cells. It may be a novel prognostic biomarker for HCC patients.     

Echo response and clinical outcome in CRT patients

Background

Change in left ventricular end-systolic volume (∆LVESV) is the most frequently used surrogate marker in measuring response to cardiac resynchronisation therapy (CRT). We investigated whether ∆LVESV is the best measure to discriminate between a favourable and unfavourable outcome and whether this is equally applicable to non-ischaemic and ischaemic cardiomyopathy.

Methods

205 CRT patients (age 65 ± 12 years, 69 % men) were included. At baseline and 6 months echocardiographic studies, exercise testing and laboratory measurements were performed. CRT response was assessed by: ∆LVESV, ∆LV ejection fraction (LVEF), ∆ interventricular mechanical delay, ∆VO₂ peak, ∆VE/VCO₂, ∆BNP, ∆creatinine, ∆NYHA, and ∆QRS. These were correlated to the occurrence of major adverse cardiac events (MACE) between 6 and 24 months.

Results

MACE occurred in 19 % of the patients (non-ischaemic: 13 %, ischaemic: 24 %). ∆LVESV remained the only surrogate marker for CRT response for the total population and patients with non-ischaemic cardiomyopathy, showing areas under the curve (AUC) of 0.69 and 0.850, respectively. For ischaemic cardiomyopathy, ∆BNP was the best surrogate marker showing an AUC of 0.66.

Conclusion

∆LVESV is an excellent surrogate marker measuring CRT response concerning long-term outcome for non-ischaemic cardiomyopathy. ∆LVESV is not suitable for ischaemic cardiomyopathy in which measuring CRT response remains difficult.     

NDRG2基因启动子甲基化与肺腺癌细胞中mRNA表达相关性的实验研究

目的:探讨肺腺癌细胞中NDRG2基因启动子甲基化状态及其与基因表达的关系。方法:甲基化焦磷酸测序技术检测启动子区域甲基化状态,荧光定量PCR技术检测不同药物浓度下培养细胞中NDRG2基因mRNA的表达水平,分析启动子区域甲基化与基因表达之间的关系。结果:在体外培养细胞中检测到NDRG2基因启动子区域呈现不同程度的甲基化,甲基化频率分别为肺癌A549细胞71.8%、GLC-82细胞86.1%、人脐静脉内皮ECV-304细胞36.8%、胃上皮GES-1细胞42.9%。NDRG2基因mRNA表达与其启动子甲基化程度成反比,甲基转移酶抑制剂5-杂氮-2-脱氧胞苷(5-Aza-CdR)作用于细胞后,A549和GLC-82细胞中NDRG2基因的mRNA转录明显上调,至72 h差异显著(P0.05)。结论:肺腺癌细胞中NDRG2基因启动子CpG岛存在高甲基化,甲基化程度与该基因的表达具有负相关性,5-Aza-CdR能在一定程度上提高NDRG2的转录水平。     

RASSF1A and DOK1 Promoter Methylation Levels in Hepatocellular Carcinoma,Cirrhotic and Non-Cirrhotic Liver,and Correlation with Liver Cancer in Brazilian Patients

Hepatocellular carcinoma (HCC) is the second most common cause of cancer mortality worldwide. Most cases of HCC are associated with cirrhosis related to chronic hepatitis B virus or hepatitis C virus infections. Hypermethylation of promoter regions is the main epigenetic mechanism of gene silencing and has been involved in HCC development. The aim of this study was to determine whether aberrant methylation of RASSF1A and DOK1 gene promoters is associated with the progression of liver disease in Brazilian patients. Methylation levels were measured by pyrosequencing in 41 (20 HCC, 9 cirrhotic, and 12 non-cirrhotic) liver tissue samples. Mean rates of methylation in RASSF1A and DOK1 were 16.2% and 12.0% in non-cirrhotic, 26.1% and 19.6% in cirrhotic, and 59.1% and 56.0% in HCC tissues, respectively, showing a gradual increase according to the progression of the disease, with significantly higher levels in tumor tissues. In addition, hypermethylation of RASSF1A and DOK1 was found in the vast majority (88%) of the HCC cases. Interestingly, DOK1 methylation levels in HCC samples were significantly higher in the group of younger (<40 years) patients, and higher in moderately differentiated than in poorly differentiated tumors (p < 0.05). Our results reinforce the hypothesis that hypermethylation of RASSF1A and DOK1 contributes to hepatocarcinogenesis and is associated to clinicopathological characteristics. RASSF1A and DOK1 promoter hypermethylation may be a valuable biomarker for early diagnosis of HCC and a potential molecular target for epigenetic-based therapy.     

Elevated Klotho Promoter Methylation Is Associated with Severity of Chronic Kidney Disease

Klotho (KL) expression is down-regulated in the renal tissues of chronic kidney disease (CKD) animal models and patients with end-stage renal disease. The putative role of KL promoter hypermethylation in the progression of CKD remains unclear. The present study aimed to determine renal and peripheral blood mononuclear cells (PBMC) levels of KL promoter methylation and analyze their relationship with clinical and histological severity in patients with CKD. Using bisulfite pyrosequencing, renal and PBMC levels of KL promoter methylation were quantified in 47 patients with CKD. 47 nephrectomy specimens of patients with renal cell carcinoma and 48 PBMC specimens of healthy volunteers were used as renal tissue and PBMC controls, respectively. Renal expression of KL protein was assayed by immunohistochemistry staining. Receiver operating characteristic (ROC) curve was used to identify the optimal cut-off value of PBMC KL promoter methylation level for renal KL promoter hypermethylation. Higher levels of KL promoter methylation were observed in renal tissue and PBMC in patients with CKD compared with controls (8.79±3.24 vs. 5.17±1.11%, P<0.001; 7.20±2.79 vs. 3.27±0.79%, P<0.001). In these patients, renal KL methylation level correlated inversely with renal KL immunostaining intensity (ρ=-0.794, P<0.001). Estimated glomerular filtration rate correlated inversely with renal and PBMC levels of KL promoter methylation (r=-0.829, P<0.001; r=-0.645, P<0.001), while tubulointerstistial fibrosis score correlated positively (ρ=0.826, P<0.001; ρ=0.755, P<0.001). PBMC KL promoter methylation level correlated positively with renal KL promoter methylation level in patients with CKD (r=0.787, P<0.001). In ROC curve, the area under curve was 0.964 (P<0.001) and the optimal cut-off value was 5.83% with a sensitivity of 93.8% and specificity of 86.7% to predict renal KL promoter hypermethylation. The degree of KL promoter methylation is associated with clinical and histological severity of CKD. PBMC KL promoter methylation level may act as a potential biomarker of renal KL promoter hypermethylation.     

Epigenetic inactivation of the metastasis suppressor RECK enhances invasion of human colon cancer cells

Down-regulation of RECK, an important metastasis suppressor gene, has been found in human colon cancer. However, the molecular mechanism for this down- regulation and its biological significance are still unclear. In the present study, we investigated whether down-regulation of RECK is caused by epigenetic inactivation via promoter methylation and tested the effect of DNA methyltransferase (DNMT) inhibitor on RECK expression and cell invasion. The mRNA and protein levels of RECK in colon tumor tissues and their normal counterparts were compared. We found that down-regulation of RECK was found in 48% of the twenty five tumors analyzed. MSP analysis demonstrated that methylation of RECK promoter was detected in 44% (11/25) of the tumor tissues and a strong correlation between down-regulation and promoter methylation was found (P = 0.028). Promoter methylation was also found in SW480 and SW620 human colon cancer cell lines. DNA methyltransferase (DNMT) inhibitor 5'-azacytidine reversed promoter methylation, restored RECK expression and suppressed invasion by these two cell lines. Restoration of RECK is critical for 5'-azacytidine-mediated suppression of cell invasion because inhibition of RECK by a specific antibody significantly attenuated the anti-invasive ability of 5'-azacytidine. Taken together, our results suggest that down-regulation of the metastasis suppressor RECK in colon cancer is associated with promoter methylation and that a DNMT inhibitor may restore RECK expression to inhibit cell invasion.     

Inactivation of Enteric Adenovirus and Feline Calicivirus by Chlorine Dioxide

Chlorine dioxide (ClO₂) inactivation experiments were conducted with adenovirus type 40 (AD40) and feline calicivirus (FCV). Experiments were carried out in buffered, disinfectant demand-free water under high- and low-pH and -temperature conditions. Ct values (the concentration of ClO₂ multiplied by contact time with the virus) were calculated directly from bench-scale experiments and from application of the efficiency factor Hom (EFH) model. AD40 Ct ranges for 4-log inactivation (Ct_99.99%) at 5°C were >0.77 to <1.53 mg/liter × min and >0.80 to <1.59 mg/liter × min for pH 6 and 8, respectively. For 15°C AD40 experiments, >0.49 to <0.74 mg/liter × min and <0.12 mg/liter × min Ct_99.99% ranges were observed for pH 6 and 8, respectively. FCV Ct_99.99% ranges for 5°C experiments were >20.20 to <30.30 mg/liter × min and >0.68 mg/liter × min for pH 6 and 8, respectively. For 15°C FCV experiments, Ct_99.99% ranges were >4.20 to <6.72 and <0.18 mg/liter × min for pH 6 and 8, respectively. Viral inactivation was higher at pH 8 than at pH 6 and at 15°C than at 5°C. Comparison of Ct values and inactivation curves demonstrated that the EFH model described bench-scale experiment data very well. Observed bench-scale Ct_99.99% ranges and EFH model Ct_99.99% values demonstrated that FCV is more resistant to ClO₂ than AD40 for the conditions studied. U.S. Environmental Protection Agency guidance manual Ct_99.99% values are higher than Ct_99.99% values calculated from bench-scale experiments and from EFH model application.     

Epigenetic inactivation of SLIT2 in human hepatocellular carcinomas

Recent findings have shown that SLIT2 appears to function as a novel tumor suppressor gene. In addition, hypermethylation of its promoter region has been detected in various cancers, including breast and lung cancer, colorectal carcinoma, and gliomas. Here, we report for the first time that there is epigenetic silencing of SLIT2 in human hepatocellular carcinoma (HCC). Downregulation of SLIT2 was detected in 6 of 8 (75%) HCC cell lines by quantitative real-time RT-PCR (qRT-PCR), and the downregulation of SLIT2 was generally dependent on the degree of methylation at the promoter region. Furthermore, expression of SLIT2 was restored in relatively low-expressing cell lines after treatment with 5-aza-2-deoxycytidine (5-Aza-dC). Downregulation of SLIT2 expression was also detected in 45 of 54 primary HCC samples (83.3%), and the decrease in expression was significantly correlated with CpG island hypermethylation. This decrease of SLIT2 expression was also associated with lymph node metastasis in HCC. Moreover, overexpression of SLIT2 in SMMC-7721 cells induced by recombinant adenovirus suppressed cell growth, migration, and invasion, These results suggest that epigenetic inactivation of SLIT2 in HCC may be important in the development and progression of HCC. Thus, SLIT2 may be useful as a therapeutic target in the treatment of HCC.     

A 3' UTR SNP in COL18A1 is associated with susceptibility to HBV related hepatocellular carcinoma in Chinese: three independent case-control studies

Background

Accumulated evidences indicate that single nucleotide polymorphisms (SNP) in angiogenesis and tumorigenesis related genes are associated with risk of Hepatocellular carcinoma (HCC). COL18A1 encodes the precursor of endostatin, which is a broad-spectrum angiogenesis inhibitor, and we speculate that SNPs in COL18A1 may be associated with susceptibility to HCC.

Methods and Findings

We carried out a 2-stage association study in 3 independent case-control groups in a total of 1067 chronic hepatitis B (CHB) patients and 808 hepatitis B virus (HBV) related HCC patients in Han Chinese. Four SNPs which can represent all potential functional SNPs with MAF>0.1 recorded in HapMap database were genotyped using TaqMan methods. Levels of total COL18A1 mRNA were also examined using quantitative real-time RT-PCR. We found that rs7499 located in 3′-UTR to be strongly associated with HBV related HCC (P_combined = 0.0000005, OR = 0.72, 95%CI = 0.63–0.82). COL18A1 mRNA expression was significantly decreased as the disease progressed (P = 0.000026).

Conclusion

These findings indicate that COL18A1 rs7499 may contribute to the risk of HCC in Han Chinese.     

巢式甲基化特异性PCR检测肝细胞癌患者血清中RASSF1A和CDH13基因启动子区甲基化

为了检测肝细胞癌患者血清中RASSF1A和CDH13基因启动子的甲基化状态,收集肝细胞癌患者及健康对照者的血清标本,采用巢式甲基化特异性PCR(nMSP)法检测RASSF1A和CDH13基因启动子区甲基化状态.结果肝细胞癌患者血清样品中RASSF1A和CDH13基因启动子区甲基化率为53.12%和31.25%,68.75%的患者血清可以检测到异常甲基化,正常对照血清中未检测到RASSF1A和CDH13基因启动子区甲基化,RASSF1A和CDH13基因甲基化与患者的临床病理资料无明显相关性(P>0.05);表明nMSP法检测血清中RASSF1A和CDH13基因启动子区甲基化具有较高的敏感性,可为肝细胞癌的筛查、早期诊断和预后判断提供有价值的信息.