Inhibition effect of miR-577 on hepatocellular carcinoma cell growth via targeting ß-catenin.

OBJECTIVE: To investigate the expression and the regulation effect of cell growth of microRNA-577 in hepatocellular carcinoma (HCC). METHODS: qRT-PCR was applied to detect the relative expression of miR-577 in 70 paired HCC and matched tumor adjacent tissues collecting from resection between March 2011 and March 2014. Pearson chi-square test was used to analyze the relationship between the miR-577 expression and clinical features. The miR-577 mimics were transfected into HepG2 cells; cell cycles were detected by flow cytometry, cell proliferation was measured by MTT assay and BrdU incorporation assay, and cell apoptosis was determined by flow cytometry and caspase3/7 activity analysis. The expressions of ß-catenin were measured by immunohistochemistry. Spearman correlation analysis was used to analyze the relationship between miR-577 and ß-catenin. qRT-PCR and western-blot were used to detect the expression of ß-catenin in transfected HepG2 cells. RESULTS: The relative expressions of miR-577 was significantly lower in HCC tissues compared to the matched normal tumor-adjacent tissues (P < 0.05). Low expression of miR-577 was significantly associated with large tumor size (≥5 cm, P < 0.05) and advanced tumor node metastasis stage (III+IV, P < 0.05). Transfection of miR-577 mimics could inhibit repress cell proliferation, enhance cell apoptosis and block the cell cycles in G0/G1 phase (P < 0.05). miR-577 in HCC group had a significant negative correlation relationship with the expression of downstream target of ß-catenin (P < 0.05). Both the mRNA and protein expression in HepG2 cells were down-regulated after transfection (P < 0.05). CONCLUSIONS: Low expression of miR-577 is related to the malignant clinicopathological features in HCC tissues, and miR-577 may suppress HCC growth through down-regulating ß-catenin.

Uncovering the pathogenesis and identifying novel targets of pancreatic cancer using bioinformatics approach.

Pancreatic cancer is a uniformly lethal disease that can be difficult to diagnose at its early stage. Thus, our present study aimed to explore the underlying mechanism and identify new targets for this disease. The data GSE16515, including 36 tumor and 16 normal samples were available from Gene Expression Omnibus. Differentially expressed genes (DEGs) were screened out using Robust Multichip Averaging and LIMMA package. Moreover, gene ontology and pathway enrichment analyses were performed to DEGs. Followed with protein-protein interaction (PPI) network construction by STRING and Cytoscape, module analysis was conducted using ClusterONE. Finally, based on PubMed, text mining about these DEGs was carried out. Total 274 up-regulated and 93 down-regulated genes were identified as the common DEGs and these genes were discovered significantly enriched in cell adhesion and extracellular region terms, as well as ECM-receptor interaction pathway. In addition, five modules were screened out from the up-regulated PPI network with none in down-regulated network. Finally, the up-regulated genes, including MIA, MET and CEACAMS, and down-regulated genes, such as FGF, INS and LAPP, had the most references in text mining analysis. Our findings demonstrate that the up- and down-regulated genes play important roles in pancreatic cancer development and might be new targets for the therapy.

Construction of pancreatic cancer double-factor regulatory network based on chip data on the transcriptional level.

Transcription factor (TF) and microRNA (miRNA) have been discovered playing crucial roles in cancer development. However, the effect of TFs and miRNAs in pancreatic cancer pathogenesis remains vague. We attempted to reveal the possible mechanism of pancreatic cancer based on transcription level. Using GSE16515 datasets downloaded from gene expression omnibus database, we first identified the differentially expressed genes (DEGs) in pancreatic cancer by the limma package in R. Then the DEGs were mapped into DAVID to conduct the kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis. TFs and miRNAs that DEGs significantly enriched were identified by Fisher's test, and then the pancreatic cancer double-factor regulatory network was constructed. In our study, total 1117 DEGs were identified and they significantly enriched in 4 KEGG pathways. A double-factor regulatory network was established, including 29 DEGs, 24 TFs, 25 miRNAs. In the network, LAMC2, BRIP1 and miR155 were identified which may be involved in pancreatic cancer development. In conclusion, the double-factor regulatory network was found to play an important role in pancreatic cancer progression and our results shed new light on the molecular mechanism of pancreatic cancer.

[Study on the etiology of acute hepatitis hospitalized patients in Beijing Ditan Hospital from 2002 to 2011].

OBJECTIVE: To explore the etiology of acute hepatitis hospitalized patients in Beijing Ditan Hospital from 2002 to 2011. METHODS: We summed up the changes in the characteristics of the etiology of acute hepatitis of patients mentioned above, and preliminarily analyze the causes. RESULTS: From 2002 to 2011, 6235 patients with acute hepatitis were admitted to Ditan Hospital, aged between 12 and 78 years old, Of which 4309 were male and 1926 female. Acute viral hepatitis accounted for 70.44%-85.07%, while CMV, EBV, drug-induced liver injury accounted less than 5%, and acute hepatitis D and acute hepatitis C less than 1.10%. From year to year, the incidence and constitution of acute hepatitis changed significantly. The proportion of patients with acute hepatitis in total hospitalized patients was from 20. 38% to 2.05%. In 10 years, the percentage of acute hepatitis A decreased most obviously, about 99.11%, while 45.07% decline in incidence of acute hepatitis B and 62. 28% of acute hepatitis E. The constituent ratio of acute hepatitis also changed significantly. The proportion of acute hepatitis A declined from 31.31% in 2002, to less than 1% in 2011. The proportion of acute hepatitis B increased from 26.47% in 2002 to 45.88% in 2011, an increase of about 2 folds in 10 years. The proportion of acute hepatitis E increased from 26.73% in 2002 to 32.05% in 2010, a rise of 1.20 times in 10 years. CONCLUSIONS: The proportion of patients with acute hepatitis in total hospitalized patients decreased from 20. 38% in 2002 to 2. 05% in 2011 in Beijing Ditan Hospital. The constituent ratio of acute hepatitis changed, too.

[Analyse related factors of impact and prognosis of 73 cases of severe hepatitis].

OBJECTIVE: A retrospective study was conducted to investigate the clinical features and prognostic factors of 73 cases of severe hepatitis. METHODS: To summarize clinical features of 73 cases of severe hepatitis, grouping by etiology and pathogenesis. A retrospective analysis was performed to evaluate the relationship between biochemical characteristics (liver function, renal function, electrolytes, PTA, etc) and complications (hepatic encephalopathy, upper gastrointestinal bleeding, hepatorenal syndrome, ascites, abdominal infections, etc) and prognosis. RESULTS: (1) HBV infection alone accounted for 65.75%. Alcoholic liver disease, drug-induced liver injury, hepatitis E, autoimmune hepatitis, overlapping causes and other factors were five cases (6.85%), six cases (8.22%), two cases (2.74%), two cases (2.74%), seven cases (9.59%) and three cases (4.11%) respectively. According to the incidence rate, severity and underlying liver condition, subacute hepatitis, cases based on chronic hepatitis and on cirrhosis were 12 cases (16.43%), 11 cases (15.07%), 50 cases (68.49%) respectively. Clinical manifestations with or without hepatic encephalopathy accounted for 58.90% or 41.10%. (2) The highest mortality of severe hepatitis was alcoholic liver disease and patients on the basis of overlapping factors (66.67%), followed by autoimmune liver disease (50%). The mortality of HBV-related hepatitis was 18.75%. Overall mortality of 73 cases of severe hepatitis was 28.77%, of which cirrhosis group was higher than non-cirrhotic group (40% vs 4.3%, P = 0.002). The difference was statistically significant. Patients without hepatic encephalopathy had lower mortality than with hepatic encephalopathy (3.33% vs 46.51%). The mortality of patients with hepatic encephalopathy Stage III and IV was 72.73%. (3) Independent samples t test filtered nine factors associated with death, namely cirrhosis, upper gastrointestinal bleeding, hepatic encephalopathy, hepatorenal syndrome, serum creatinine, total bilirubin (TBIL), direct bilirubin (DBIL), albumin (ALB) and serum sodium. The results of multivariate conditional logistic regression analysis indicated that hepatic encephalopathy, serum creatinine levels were risk factors for death, whereas ALB as a protective factor. CONCLUSION: Hepatic encephalopathy, serum creatinine levels were risk factors for severe hepatitis death, But ALB was protective factor. Nucleotide analogs using was the main reason why the mortality of hepatitis B was as low as 18.75%.

Multivariate prognostic study on node-positive gastric cancer: is tumor size a prognostic indicator?

BACKGROUND/AIMS: We analyzed the clinicopathological factors of patients with node-positive gastric cancer, evaluated the prognostic factors associated with long-term survival and clarified the effect of tumor size on long-term survival. METHODOLOGY: The study included 591 patients who underwent curative resection for node-positive gastric cancer. Clinicopathological prognostic variables were evaluated as predictors of long-term survival by univariate and multivariate analyses. RESULTS: The 5-year survival rate was influenced by tumor size, tumor location, depth on invasion, level of lymph node metastasis, Borrmann classification, histological type, liver metastasis, peritoneal dissemination and disease stage. Of these, independent prognostic factors were depth on invasion and lymph node metastasis. Tumor size is an influence but not independent factor for the prediction of long-term survival in patients with node-positive gastric cancer. CONCLUSIONS: In patients with node-positive gastric cancer, two independent prognostic factors were depth on invasion and the status of lymph node metastasis.

[Study on effect of intensive treatment for refractory chronic hepatitis C patients].

OBJECTIVE: To explore the effect of intensive treatment for refractory chronic hepatitis C, and to improve the sustained viral response (SVR) rate of treatment with interferon plus ribavirin by optimizing therapeutic dose and course. METHODS: Patients who did not acquire response or partial response by standard therapy (PEG-IFN alpha subcutaneous injection weekly plus Ribavirin 10.5 mg/kg) every day were enrolled and retreated with intensive treatment of 10 MU interferon every other day or 360 microg pegylated interferon alpha-2a weekly according to patients' wishes, and ribavirin 15 mg/kg every day. Serum HCV RNA was detected at baseline,treatment week 4, 12 and every 12 weeks succedent and 24 weeks after treatment end. Course of treatment was 72 to 96 weeks according to viral response. SVR was the mark of therapeutic effect. RESULTS: 18 patients completed whole range therapy and follow-up, in which 12 patients acquired SVR, 5 patients treatment failure and 1 relapse. 3 patients acquired rapid viral response (RVR), and they all got complete Early Viral Response (cEVR) and SVR. RVR Patients' viral loads were significantly lower than that of patients who did not acquire RVR (t = 4. 687, P < 0.001). In 15 patients who did not acquire RVR, 8 patients acquired cEVR, and 9 acquired SVR. SVR rate of patients who were administered PEG-IFN alpha-2a was 4/5, 11 patients who acquired cEVR all acquired SVR, while in 7 patients who did not acquire cEVR, only 1 patient acquired SVR. CONCLUSIONS: High percent patients, who did not acquire response or partial response by previous standard antiviral therapy, could gain SVR by intensive dose interferon plus Ribavirin. In intensive treatment procedure, adjusting and prolonging course according to viral response after HCV RNA turned negative were important measures to improve refractory Chronic Hepatitis C SVR rate.

[Efficacy of antiviral treatment on intrahepatic HBV DNA and histology in HBeAg -positive chronic hepatitis B patients].

OBJECTIVE: To evaluate the effect of antiviral agents on intrahepatic HBV DNA and histology in HBeAg-positive chronic hepatitis B patients. METHODS: Thirty-five patients were treated with lamivudine, 16 with interferon alfa (INF-alpha), 24 with sequential Lamivudine and INF-alpha. The total duration of therapy was 12 months. Intrahepatic HBV DNA was measured quantitatively by real-time polymerase chain reaction. RESULTS: There was significant change in all parameters of the groups of patients at the end of treatment (P < 0.05). The patients treated with sequential treatment had slightly higher HBeAg seroconversion rate (38.1%) than that of the other patients (P=0.1352). The baseline levels of intrahepatic HBV DNA in the patients with HBeAg seroconversion or undetectable serum HBV DNA were significantly lower than that of the other patients (P < 0.05). CONCLUSION: Antiviral agents could effectively inhibit intrahepatic HBV DNA and improve hepatic histology. The patients with low baseline intrahepatic HBV DNA level may achieve better antiviral efficacy. Sequential treatment might produce high HBeAg seroconversion rate.

[Effects of antiviral agents on intrahepatic ccc DNA in HBeAg-positive chronic hepatitis B patients].

OBJECTIVE: To evaluate the effects of antiviral agents on intrahepatic HBV covalently closed circular DNA (cccDNA) in HBeAg-positive chronic hepatitis B patients. METHODS: Seventy-one HBeAg positive chronic hepatitis B patients were enrolled in this study. Lamivudine was administered to 35 patients for 48 weeks, sequential therapy with lamivudine-IFN alpha-2b to 24 of the 71 patients for 48 weeks, and interferon alpha (IFN alpha-2b) was administered to 12 for 24 weeks. All subjects were followed-up for 24 weeks. Serum HBV DNA, intrahepatic HBV DNA and cccDNA were measured quantitatively by PCR. HBV genotypes were analyzed by PCR-RFLP. RESULTS: Forty-eight weeks of sequential lamivudine-IFN alpha-therapy and lamivudine monotherapy and 24 weeks of IFN alpha monotherapy reduced the intrahepatic HBV DNA to (4.7+/-1.1) log10, (4.6+/-1.5) log10 and (5.6+/-1.5) log10, and cccDNA to (3.4+/-1.3) log10, (3.8+/-1.1) log10 and (5.0+/-1.5) log10, significantly lower than therapy (P < 0.05). Seventeen of the 71 patients developed HBeAg seroconversion, and the reduction of cccDNA in the HBeAg seroconverted patients was significantly more than that of the HBeAg positive patients (P < 0.05). After 24 weeks of antiviral therapy withdrawal, 18 patients achieved sustained virological response, and the baseline intrahepatic cccDNA in the patients with sustained virological response was significantly lower than that of patients with virological rebound (P < 0.05). The change in intrahepatic cccDNA correlated positively with the reduction in intrahepatic HBV DNA (P < 0.05). The cccDNA levels correlated with the serum HBeAg titers at the end of the treatment (P < 0.01). Of the total 71 cases, HBV genotype C accounted for 85.9% (n = 61), and genotype B for 14.1% (n = 10). There was no significant difference in the changes of intrahepatic HBV DNA and cccDNA levels between HBV genotypes C and B (P >0.05). CONCLUSIONS: Both 48 weeks of sequential lamivudine-IFN alpha and lamivudine monotherapy strongly reduced intrahepatic HBV DNA and cccDNA more than 24 weeks of IFN alpha monotherapy. Low baseline intrahepatic cccDNA levels might predict a good long-term efficacy of antiviral treatment. The reduction of intrahepatic cccDNA correlated positively with the changes of intrahepatic HBV DNA, and intrahepatic cccDNA levels correlated with serum HBeAg titers. HBV genotypes had no obvious influence on intrahepatic HBV DNA load or cccDNA load.

Effects of antiviral agents and HBV genotypes on intrahepatic covalently closed circular DNA in HBeAg-positive chronic hepatitis B patients.

AIM: To evaluate the effects of antiviral agents and HBV genotypes on intrahepatic covalently closed circular DNA (ccc DNA) in HBeAg-positive chronic hepatitis B patients. METHODS: Seventy-one patients received lamivudine (n = 35), or sequential therapy with lamivudine- interferon alpha 2b (IFN-alpha 2b, n = 24) for 48 wk, or IFN-alpha 2b (n = 12) for 24 wk. All subjects were followed up for 24 wk. Intrahepatic ccc DNA was measured quantitatively by PCR. HBV genotypes were analyzed by PCR-RFLP. RESULTS: Sequential lamivudine- INF-alpha therapy, lamivudine and INF-alpha monotherapy reduced ccc DNA of 1.7 log, 1.4 log and 0.8 log, respectively (P < 0.05). Seventeen out of the 71 patients developed HBeAg seroconversion, the reduction of ccc DNA in the HBeAg seroconversion patients was more significant than that in the HBeAg positive patients (3.0 log vs 1.6 log, P = 0.0407). Twenty-four weeks after antiviral therapy withdrawal, 16 patients had a sustained virological response, the baseline intrahepatic ccc DNA in the patients with a sustained virological response was significantly lower than that in the patients with virological rebound (4.6 log vs 5.4 log, P = 0.0472). HBV genotype C accounted for 85.9% (n = 61), and genotype B for 14.1% (n = 10), respectively, in the 71 patients. There was no significant difference in the change of ccc DNA level between HBV genotypes C and B (2.1 log vs 1.9 log). CONCLUSION: Forty-eight week sequential lamivudine-INF-alpha therapy and lamivudine monotherapy reduce ccc DNA more significantly than 24-wk INF-alpha monotherapy. Low baseline intrahepatic ccc DNA level may predict the long-term efficacy of antiviral treatment. HBV genotypes C and B have no obvious influence on ccc DNA load.

[Establishment and application of a real-time PCR method to detect hepatitis B virus cccDNA quantitatively].

OBJECTIVE: To establish a new method to detect HBV cccDNA quantitatively and to apply it to detect cccDNA in liver needle biopsy specimens of chronic hepatitis B patients. METHODS: The sequences of HBV DNA genotypes A through G were analyzed. According to the different sequence structure of cccDNA and rcDNA, primes and probe were designed in highly conservative region outside the nick of cccDNA in order to amplify cccDNA but not rcDNA. The best conditions of this method were found after testing experiments. Also we checked its specificity and sensitivity and reproducibility. The products of PCR were sequenced in order to ascertain if it was the right region expected. To amplify with standard plasmid ranged from 10(2) to 10(10) copies/ml to measure the sensitivity and amplify in parallel with standard plasmid of 10(6) copies/ml for 30 replicates so as to measure its reproducibility. DNA was extracted from 32 needle liver biopsy specimens of chronic hepatitis B patients. The cccDNA was quantitatively detected with this method. The data of cccDNA obtained before and after therapy and their relationship with total HBV DNA were analyzed. RESULTS Results of sequencing showed that the PCR product was from the right region. The sensitivity was 10(3)-10(10) copies/ml. The Ct value was 29.69+/-0.31 and the coefficient of variability was 1.04 percent calculated from the data of 30 PCR reactions with standard plasmid. The percentage of decrease in serum HBV DNA, total HBV DNA in liver and cccDNA in liver were 0.49+/-0.17, 0.22+/-0.18 and 0.16+/-0.28 respectively. There is 47 percent-98 percent cccDNA in total HBV DNA in liver and the mean is 81.5 percent. CONCLUSION: The method is good because of the simple and convenient operation, the high specificity, the wide linear detection range and the fine reproducibility. Therefore it can be used for both scientific research and clinical purpose. Lamividine can significantly inhibit serum HBV DNA by, but its inhibitory effect on cccDNA in liver was rather weak.

Intrahepatic HBV DNA as a predictor of antivirus treatment efficacy in HBeAg-positive chronic hepatitis B patients.

AIM: To evaluate the effect of antiviral agents on intrahepatic HBV DNA in HBeAg-positive chronic hepatitis B patients. METHODS: Seventy-one patients received treatment with lamivudine, interferon alpha (IFN-alpha 2b) or sequential therapy with lamivudine-IFN-alpha 2b for 48 wk. All subjects were followed up for 24 wk. Serum and intrahepatic HBV DNA were measured quantitatively by PCR. HBV genotypes were analyzed by PCR-RFLP. RESULTS: At the end of treatment, the intrahepatic HBV DNA level in 71 patients decreased from a mean of (6.1 +/- 1.0) log10 to (4.9 +/- 1.4) log10. Further, a larger decrease was seen in the intrahepatic HBV DNA level in patients with HBeAg seroconversion. Intrahepatic HBV DNA level (before and after treatment) was not significantly affected by the patients' HBV genotype, or by the probability of virological flare after treatment. CONCLUSION: Intrahepatic HBV DNA can be effectively lowered by antiviral agents and is a significant marker for monitoring antivirus treatment. Low intrahepatic HBV DNA level may achieve better efficacy of antivirus treatment.