Ying-Tang Gao

Aberrant DNA methylation profile of hepatocellular carcinoma and surgically resected margin.

Field cancerization currently described the theory of tumorigenesis and, until now, has been described in almost all organ systems except in liver. For this reason, we explore the presence of field cancerization in liver and its underlying clinical implication in hepatocellular carcinoma (HCC). In our study, methylation profile of HCC and surgically resected margin (SRM) were established by methylation‐specific PCR. Liver cirrhosis (LC), chronic hepatitis and normal liver were treated in the same way as the background control. The correlation analysis among the methylation profile of HCC, SRM and clinicopathological data of HCC patients was made respectively. Our results showed that methylation abnormities related to HCC, but not background disease existed in histologically negative SRM. Monoclonal and polyclonal models may coexist in field cancerization in liver. Patients with RIZ1 methylation in SRM had a shorter disease free survival. The local recurrence trend of early and later recurrence in HCC is potentially related to a second field tumor. From these results, we can suggest that field cancerization exists in liver. The study of field cancerization in liver plays an important role in hepatocarcinogenesis. Second field tumor derived form field cancerization may have important implications in HCC prognosis assessment that is worthy of further study. (Cancer Sci 2009; 100: 996–1004)

Expression and significance of microRNA-183 in hepatocellular carcinoma.

Objective. In our previous study, we found that some miRNAs were deregulated in hepatocellular carcinoma (HCC), including miR-183. However, the expression of miR-183 in the progression of benign liver diseases to HCC and its correlation with clinicopathologic factors remain undefined. Methods. MiR-183 expression was measured in normal controls (NC) (n = 21), chronic viral hepatitis B or C (CH) tissues (n = 10), liver cirrhosis (LC) tissues (n = 18), HCC tissues (n = 92), and adjacent nontumor tissues (NT) (n = 92) by quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR). Results. The expression levels of miR-183 were significantly higher in HCC than in NT, LC, CH, and NL (P = 0.001, P < 0.001, P = 0.011, P < 0.001, resp.). The upregulated miR-183 in HCC was correlated with TNM stage (P = 0.042) and cirrhosis (P = 0.025). The Kaplan-Meier survival analysis showed that miR-183 expression was not associated with the survival of HCC patients. However, miR-183 yielded an area under the curve (AUC) of 0.808 with 59.8% sensitivity and 91.8% specificity in discriminating HCC from benign liver diseases (CH and LC) or NC. Conclusions. The upregulated miR-183 may associate with onset and progression of HCC, but not with the patient survival. A further research is needed to determine the potential of miR-183 as biomarker for HCC.

Enhanced specificity of real-time PCR for measurement of hepatitis B virus cccDNA using restriction endonuclease and plasmid-safe ATP-dependent DNase and selective primers

The persistence of covalently closed circular DNA (cccDNA) of hepatitis B virus (HBV) in hepatocytes plays a key role in viral persistence and resistance to therapy. Therefore, quantitative cccDNA measurement is of clinical importance for evaluating the efficacy of antiviral drugs, selecting an appropriate treatment strategy, and predicting the prognosis. Current established methods for measurement of cccDNA need further improvement. A modified method was developed using digestion with restriction endonucleases that do not recognize sites in the HBV DNA and plasmid-safe ATP-dependent DNase (PSAD), and using a cccDNA-specific primer set in a real-time PCR reaction. The cccDNA-specific primer has a similar amplification efficiency as a commercial kit. Treatment of samples with restriction endonuclease followed by PSAD digestion increased significantly the specificity of a cccDNA-selective primer set compared with other treatments (P<0.05). Analysis of 35 serum and liver DNA samples from patients with hepatocellular carcinoma demonstrated that the amount of serum cccDNA is beyond the minimum detection limit and that the liver cccDNA quantity is about 0-49.2 copies/cell, consistent with previous reports. Taken together, this method has the potential for evaluating the efficacy of antiviral drugs.

Expression and Clinical Significance of Livin Protein in Hepatocellular Carcinoma

In this study, the two-step PV method of immunohistochemistry was used to determine livin protein expression in HCC tissues, pericarcinoma tissues, hepatitis/hepatic cirrhosis tissues, and normal hepatic tissues, and livin protein expression was detected in the blood plasma of patients with HCC before and after surgery, subjects with hepatic cirrhosis and hepatitis, and healthy blood donors using ELISA. Livin protein expression was significantly higher in HCC tissues than that in normal hepatic tissues and hepatitis/hepatic cirrhosis tissues, with no significant difference between HCC tissues and pericarcinoma tissues. The HCC patients with positive livin protein expression had a significantly higher survival rate than those with negative livin protein expression. Livin protein expression was significantly higher in the blood plasma of patients with HCC before and after surgery and in patients with hepatic cirrhosis and hepatitis than that in healthy blood donors, whereas livin protein expression in the blood plasma of patients with HCC was not significantly different from that of patients with hepatic cirrhosis and hepatitis. Livin protein expression in HCC tissues did not correlate with that in the blood plasma of the same HCC patients. Livin protein expression may be a potential, effective indicator for assessing prognosis in patients with HCC.

Overexpression of microRNA let-7 correlates with disease progression and poor prognosis in hepatocellular carcinoma

Abstract The aim of the study was to explore the clinical significance of let-7 expression in hepatocellular carcinoma (HCC). A PCR array was conducted to screen for let-7 expression in early-stage HCC. Next, the deregulation of let-7 was confirmed by quantitative real-time RT-PCR (qRT-PCR) in another set of liver tissues, including normal control (NC), chronic hepatitis (CH), liver cirrhosis (LC), HCC, and adjacent nontumor (NT) tissues. In addition, as the potential target mRNA of let-7, alpha 2(I) collagen (COL1A2) mRNA was also quantified in the above liver tissues. Finally, an association study comparing let-7 and COL1A2 and their clinical significance in HCC was conducted. PCR array analysis revealed that the expression levels of let-7a/7b/7c were significantly downregulated in early-stage HCC compared to those in NT tissues. As compared to NC samples, qRT-PCR further confirmed that let-7a/7b/7c/7e were significantly upregulated in CH, LC, and NT tissues, while there were no significant differences in expression between the HCC and NC groups. Although COL1A2 may be the target mRNA of let-7, only let-7c expression was inversely correlated with COL1A2 mRNA expression in CH tissues. In HCC tissues, levels of let-7a/7b/7c/7e were positively correlated with that of COL1A2 mRNA. The clinical significance study revealed that elevated let-7a expression was significantly correlated with serosal and vein invasion, while elevated let-7c expression was significantly correlated with vein invasion and advanced TNM stage. Elevated let-7e expression was significantly correlated with vein invasion in HCC. Significantly shorter postoperative overall survival was observed in HCC patients with high let-7c expression. The results suggest that aberrant expression of let-7a/7b/7c/7e occurs in benign liver diseases and HCC. The upregulation of let-7 expression is associated with the progression and poor prognosis of HCC, and further mechanistic studies are warranted.

Identification and validation of specific methylation profile in bile for differential diagnosis of malignant biliary stricture

OBJECTIVE This study was aimed to identify the specific methylation profile in bile specimens of pancreaticobillary diseases for differential diagnosis of malignant biliary stricture. DESIGN AND METHODS In a total of 80 bile specimens from pancreaticobillary diseases, the methylation status of 19 tumor suppressor genes were analyzed by methylation-specific PCR and the methylation index (MI) were compared between the malignant and benign groups. RESULTS Methylation of DKK3, p16, SFRP2, DKK2, NPTX2 and ppENK were more frequently detected in the bile of malignant biliary strictures than benign patients. When setting MI 0.5 as the threshold, this 6-gene panel could distinguish the malignant biliary stricture with a high sensitivity, specificity and accuracy (77.27%, 77.78% and 77.50%, respectively). CONCLUSION The methylation profile including 6 specific genes in bile may be a promising biomarker for differential diagnosis between malignant and benign biliary strictures.

The expression of thymosin β4 in chronic hepatitis B combined nonalcoholic fatty liver disease.

AbstractThe aim of the study was to detect the expression level of thymosin &bgr;4 (T&bgr;4) in serum and tissues of patients with chronic hepatitis B (CHB) combined nonalcoholic fatty liver disease (NAFLD). The effects of T&bgr;4 in hepatic steatosis, chronic inflammation, and fibrosis development in CHB combined NAFLD patients were also discussed. The study included 46 patients in the case group with CHB and NAFLD and 42 patients in the control group with CHB. ELISA was applied to detect serum T&bgr;4 and TNF-&agr; level. Furthermore, the correlation analysis of T&bgr;4 levels with biochemical index, pathological index, and TNF-&agr; level was performed. The T&bgr;4 immunohistochemical levels of different inflammation fibrosis levels were compared, and the correlation analysis with TNF expression was performed. The T&bgr;4 levels in patients with CHB combined NAFLD showed no statistical difference when compared to the control group. In patients with CHB combined NAFLD group, the T&bgr;4 level had no correlation with ALT, AST, TG, FGP, hepatitis B virus (HBV)-DNA levels, and fat grading, but had negative correlation with inflammation score and fibrosis score (P <0.01). The immunohistochemical results of hepatic tissues showed that the expression intensity of severe inflammation fibrosis group had statistical significance compared with that of slight group, and the T&bgr;4 expression both in serum and in liver tissue negatively correlated with TNF-&agr; expression. T&bgr;4 could be involved in the regulation of chronic inflammation and fibrosis and plays a defense role in the disease progression of CHB combined NAFLD patients.

Real-time live-cell analysis system for screening single tumor cell clones and analyzing their colony-forming ability

AIM To screen single tumor cell clones and evaluating their colony-forming ability by ncuCyte ZOOM. METHODS Primary tumor cells were isolated by differential digestion and differential adherence method. On the basis of limited dilution, dynamic realtime tracking technology and full aperture imaging technology were used to track single cell clones and evaluate their colony-formation ability. RESULTS Six lines of primary tumor cells (TJ3ZX-02 实时动态活细胞成像系统在单克隆筛选和克隆形成能力 检测中的应用 刘长政, 焦晓磊, 高敦芹, 邢龙彬, 刘 辉, 骆 莹, 高英堂 在线投稿: http://www.baishideng.com/wcjd/ch/index.aspx DO : 10.11569/wcjd.v25.i10.881 世界华人消化杂志 2017年4月8日; 25(10): 881-890 SSN 1009-3079 (print) SSN 2219-2859 (online) 基础研究 BASIC RESEARCH ® ■同行评议者 陈绍勤, 副教授, 主任医师 , 福建 医科大学附属第 一医院胃肠外科 二病区; 韩双印, 主任医师 , 郑州 大学人民医院消 化内科; 李云龙, 副教授 , 哈尔滨 医科大学附属二 院普通外科 ; 王 雅棣, 教授, 主任 医师 , 北京军区 总医院放疗科 2017-04-08|Volume 25| ssue 10| WCJD|www.wjgnet.com 881 冻存; 并且通过时序图准确有效地排除了 18个不能持续增殖的单克隆和28个由2个 及以上细胞组成的多克隆 . 2例单克隆株 (TJ3ZX-06-B11, TJ3ZX-07-H11)克隆形成能 力分析表明, 14 d时平皿方法的克隆形成率 (35.17%, 13.17%)高于IncuCyte ZOOM 96孔 全孔成像(23.13%, 5.51%), 且差异有统计学 意义(P <0.05); 延长至21 d时全孔成像的克隆 形成率为(35.63%, 13.22%), 与平皿方法比较 无统计学差异. 结论 IncuCyte ZOOM系统能够简便、准确、省时 省力地实现单克隆细胞筛选和克隆形成率 检测.

The MTHFR C677T mutation is not a risk factor recognized for HBV-related HCC in a population with a high prevalence of this genetic marker.

BACKGROUND Polymorphism of the methylenetetrahydrofolate reductase (MTHFR) gene can affect disease progression in HBV infection. However, the results from different reports are inconsistent. The aim of this study was to investigate the association between the MTHFR C677T polymorphism and the outcome of HBV infection in a Tianjin Han population. METHODS TaqMan SNP genotyping was employed to determine the alleles and genotypes of MTHFR C677T in 2511 subjects from various stages of HBV infection and 549 healthy controls. RESULTS Of the 3060 subjects, the genotypic frequencies were CT 48.9%, TT 29.3% and CC 21.8%; the allelic frequencies were T 53.8% and C 46.2%. There was no significant difference in genotypic or allelic distribution among the different disease groups. When either healthy subjects or self-limited subjects were used as controls, the TT genotype and the T allele conferred protective effects against hepatocellular carcinoma (HCC) (HCC vs healthy subjects: OR=0.588, 95% CI=0.413-0.836, P=0.003; OR=0.768, 95% CI=0.645-0.915, P=0.003, respectively. HCC vs self-limited subjects: OR=0.598, 95% CI=0.404-0.886, P=0.010; OR=0.772, 95% CI=0.635-0.940, P=0.010, respectively). After sub-stratification by gender, the prevalence of the TT genotype or T allele was the lowest in the male HCC group (TT 23.5%, T 49.8%). The protective effects of the TT genotype and the T allele were observed in male HCC and cirrhotic subjects (HCC vs self-limited subjects: OR=0.470, 95% CI=0.288-0.766, P=0.002; OR=0.681, 95% CI=0.535-0.866, P=0.002, respectively. Liver cirrhosis vs self-limited subjects: OR=0.624, 95% CI=0.392-0.992, P=0.046; OR=0.791, 95% CI=0.627-0.998, P=0.048, respectively), but not in female. When the subjects were stratified according to the clinical features, no statistically significant difference in the genotypic distribution was observed (P>0.05). CONCLUSIONS The TT genotype and T allele of MTHFR C677T may confer a protective effect on disease progression to HCC in HBV-infected individuals, especially among male patients, in a population with a high prevalence of this genetic marker.

A Noninvasive Score Model for Prediction of NASH in Patients with Chronic Hepatitis B and Nonalcoholic Fatty Liver Disease

Aims. To develop a noninvasive score model to predict NASH in patients with combined CHB and NAFLD. Objective and Methods. 65 CHB patients with NAFLD were divided into NASH group (34 patients) and non-NASH group (31 patients) according to the NAS score. Biochemical indexes, liver stiffness, and Controlled Attenuation Parameter (CAP) were determined. Data in the two groups were compared and subjected to multivariate analysis, to establish a score model for the prediction of NASH. Results. In the NASH group, ALT, TG, fasting blood glucose (FBG), M30 CK-18, CAP, and HBeAg positive ratio were significantly higher than in the non-NASH group (P < 0.05). Multivariate analysis showed that CK-18 M30, CAP, FBG, and HBVDNA level were independent predictors of NASH. Therefore, a new model combining CK18 M30, CAP, FBG, and HBVDNA level was established using logistic regression. The AUROC curve predicting NASH was 0.961 (95% CI: 0.920–1.00, cutoff value is 0.218), with a sensitivity of 100% and specificity of 80.6%. Conclusion. A noninvasive score model might be considered for the prediction of NASH in patients with CHB combined with NAFLD.