| PartⅠ Clinic Value of Serum Ubiquitin-Conjugating Enzyme2 Q1(UBE2Q1)Gene Methylation in Hepatocellular CarcinomaBackgroudHepatocellular carcinoma(HCC)is the most common type of liver cancer.Hepatitis B virus(HBV)infection is the main risk factor for HCC.Indeed,the five-year survival rate of HCC is approximately 5%,due to late diagnosis or recurrence and metastasis after resection.Liver biopsy is the most reliable standard for diagnosis of liver cancer,but liver biopsy is an invasive and not suitable for early screening of liver cancer.Magnetic resonance imaging and computed tomography are the most important methods for diagnosis of liver cancer,but it is easy to miss diagnosis for small diameter liver cancer.Alpha fetoprotein(AFP)is a commonly used serological marker for liver cancer screening,but its sensitivity and specificity are not high.Circulating free DNA(cfDNA)has the characteristic changes associated with tumorigenesis and development.cfDNA methylation may be a serological marker for tumor diagnosis.DNA methylation plays an important role in gene expression regulation,carcinogenesis and other important biological processes.Abnormal DNA methylation expression has potential clinical value in the diagnosis and prognosis of hepatocellular carcinoma.Ubiquitin proteasome system is an important protein degradation pathway in eukaryotic cells.The abnormality of ubiquitin proteasome system is closely related to tumor cell proliferation,apoptosis,metastasis and invasion,which can lead to tumor occurrence and development.Ubiquitin binding enzyme UBE2Q1 is overexpressed in many malignant tumors and plays the role of oncogene.However,the methylation status of the UBE2Q1 gene in the hepatocellular carcinoma is still unknown.ObjectiveThe methylation status of UBE2Q1 gene in peripheral serum cfDNA of patients with hepatitis B virus associated hepatocellular carcinoma(HBV-related HCC),hepatitis B virus associated cirrhosis patients(HBV-related LC),chronic hepatitis B(CHB)and healthy volunteers(HCs)was detected.The correlation between the methylation status of UBE2Q1 gene and clinical data in patients with HBV-related HCC was analyzed.To investigate the feasibility of methylation of UBE2Q1 gene as a serological marker in the diagnosis of hepatitis B virus associated hepatocellular carcinoma.MethodsThis study enrolled 80 patients with HBV-related HCC,40 patients with HBV-related LC and 40 patients with HBV-related from July 2015 to July 2016 at Qilu Hospital of Shandong University.20 cases of HCs were used as clinical control.The methylation rate of serum UBE2Q1 gene was detected by methylation specific PCR.The methylation frequency of UBE2Q1 gene in each group was compared by chi-square test.Chi-square test and spearman correlation analysis were used to analyze the correlation between UBE2Q1 gene methylation status and clinical parameters in HBV-related HCC patients.The ROC curve was used to analyze the values of the methylation status of UBE2Q1 gene and combined with serum AFP level in the diagnosis of HBV-related HCC.Results1.The methylation rate of serum UBE2Q1 gene in HBV-related HCC group is 33.75%(27/80),The methylation rates of serum UBE2Q1 gene in HBV-related LC group,CHB group and HCs group were 55.00%(22/40)、60.00%(24/40)and 65.00%(13/20)respectively.The methylation frequency of HBV-related HCC group was significantly lower than that of HBV-related LC group,CHB group and HCs group.There was no significant difference in methylation frequency between HBV-related LC group,CHB group and HCs group.2.The serum UBE2Q1 methylation frequency in patients with advanced TNM stage(Ⅲ-Ⅳ)was lower than that in patients with early TNM stage(Ⅰ-Ⅱ)(χ2=4.68,P =0.030).The serum UBE2Q1 methylation frequency of patients with tumor diameter greater than 3 cm was lower than that of patients with tumor diameter less than 3 cm(χ2=5.09,P = 0.024).Spearman correlation analysis showed significant negative correlation between serum UBE2Q1 methylation rate and TNM stage.Correlation coefficient rs with tumor diameter was-0.20,P=0.080.There was no significant correlation in HBV-related HCC patients between serum UBE2Q1 methylation rate and other clinical parameters,such as sex,age,HBeAg,Child-Turcotte-Pugh score and portal vein invasion.3.The sensitivity and specificity distinguishing of HCC patients from LC and CHB patients was 66.30%,57.50%by using serum UBE2Q1 methylation status,AUC 0.619,95%CI 0.532-0.706.Using serum AFP level alone,the sensitivity and specificity to distinguish HCC patients from LC and CHB patients was 53.80%,87.50%.AUC was 0.668,95%CI 0.581-0.755.The AFP cut-off value given by the ROC curve was 192.15ng/ml.We took 20ng/ml as the cut-off values for AFP positive,the sensitivity and specificity of serum UBE2Q1 methylation combined with AFP level in the diagnosis of hepatocellular carcinoma was 58.80%,75.00%.AUC was 0.720,95%CI 0.641-0.799.On this basis,we further analyze the clinic values in the diagnosis of hepatocellular carcinoma,combined with the methylation status of UBE2Q1 and AFP cut-off values 200ng/ml or 400ng/ml respectively,the sensitivity and specificity was 53.80%and 87.50%,37.50%and 88.70%,AUC was 0.760 and 0.694.Conclusions1.The abnormal expression of UBE2Q1 gene methylation in the serum of HBV-related HCC patients,combined with serum AFP level can improve the diagnostic rate of HCC,suggesting that serum UBE2Q1 gene methylation can be used as a potential marker for the detection and diagnosis of HCC.2.Abnormal UBE2Q1 methylation was negatively correlated with TNM stage in patients with HBV-related HCC,suggesting that serum UBE2Q1 gene methylation could be used to judge the prognosis of HCC patients.Part II Study on UBE2Q1 gene methylation in peripheral blood mononuclear cells in patients with acute-on-chronic hepatitis B pre-liver failureBackgroudLiver failure is a common clinical syndrome of severe liver disease with rapid progression and high mortality.Liver failure can be divided into four categories:acute liver failure(ALF),subacute liver failure(SALF),acute-on-chronic liver failure(ACLF)and chronic liver failure(ACLF).Acute-on-chronic liver failure(ACLF)is the most common type of liver failure in China.The main cause of liver failure in China is hepatitis B virus infection.If hepatitis B virus infection progresses to liver failure,treatment can be extremely difficult,and mortality is very high.The major clinical manifestations of such progression include the features of ACLF.Compared with chronic hepatitis,ALCF features acute disease onset and rapid disease progression,but its progression is slower than that of acute liver failure without chronic liver disease.To this end,there is a time window from ACLF onset to the occurrence of liver failure.This time window is called "acute-on-chronic pre-liver failure(pre-ACLF)".Early warning of the risk of HBV-related ACLF(ACHBLF)and the identification of specific markers for the diagnosis of HBV-related ACLF have important clinical significance.At present,there are three different definitions of liver failure in our country,but the treatment scheme emphasizes active support and symptomatic treatment on the basis of comprehensive medical treatment.According to studies conducted by domestic experts and researchers,the main points on which clinical diagnosis of pre-ACHBLF are based in this study are as follows:extreme fatigue,obvious gastrointestinal symptoms,including loss of appetite,vomiting and bloating;TBIL≥171μmol/l;PTA>40%.Apoptosis and necrosis of hepatocytes,inflammatory cell infiltration and ischemic liver damage are considered the key factors in the pathogenesis of liver failure.Immune-mediated liver injury is involved too.The ubiquitin-proteasome system(UPS)is a major protein control system in eukaryotic cells and participates in many physiological processes in the body,including cell proliferation,differentiation,apoptosis,DNA damage repair and immune response.The UPS plays an important role in the regulation of inflammatory responses and can promote tissue inflammatory responses by activating the NF-kB signaling pathway.The alteration of proteasome activity can regulate the inflammatory response which also plays an important role in patients with ACHBLF during the progression of the disease.We speculate that the UPS may participate in the progression of ACHBLF.The ubiquitin-binding enzyme E2 is one of the important members of the UPS and is closely related to cell proliferation,apoptosis,the immune response and tumorigenesis.DNA methylation is one of the most important epigenetic modifications.Studies have shown abnormal DNA methylation expression in peripheral blood mononuclear cells in patients with ACHBLF.Our previous studies showed that the glutathione-S-transferase P1(GSTP1)gene was aberrant methylated in Pre-ACHBLF.However,studies on the UBE2Q1 methylation status in Pre-ACHBLF have not been reported.ObjectiveThe methylation frequency of UBE2Q1 gene in peripheral blood mononuclear cells(PBMC)of patients with Pre-ACHBLF,patients with chronic hepatitis B(CHB)and Health controls(HCs)were detected.Correlation between methylation status of UBE2Q1 Gene and Clinical parameters in peripheral blood mononuclear cells of patients with Pre-ACHBLF and Clinical parameters of early warning of the occurrence of ACHBLF were analyzed.The aim was to explore the feasibility of UBE2Q1 gene methylation status as a biomarker of ACHBLF early prediction and prognosis.Methods60 patients with Pre-ACHBLF and 40 patients with CHB were enrolled in the department of hepatology at Qilu Hospital of Shandong University from December 2015 to July 2016.20 cases of HCs were used as clinical control.Pre-ACHBLF were followed-up.UBE2Q1 gene methylation frequency in peripheral blood mononuclear cells was detected by methylation specific PCR,Chi-square test and Spearman correlation coefficient were used to analyze the correlation between methylation status of UBE2Q1 gene and clinical parameters of pre-ACHLF patients.Cox proportional risk model was used to analyze the clinical parameters of early warning ACHBLF.The feasibility of early diagnosis of ACHBLF was detected through analyzing the values of UBE2Q1 gene methylation and combined MELD score by ROC curve.Results1.Age,gender and serum creatinine levels did not differ significantly among the three groups(P>0.001),whereas ALT,AST,TBIL,ALB,INR and PTA levels,which indicate the severity of liver failure,were significantly different.There was no significant difference in HBeAg or HBV-DNA levels between the ACHBLF and CHB groups.2.The methylation-specific PCR assay showed that the methylation rate of the UBE2Q1 gene in the peripheral blood mononuclear cells of 60 patients with pre-ACHBLF was 38.33%,significantly lower than that in the 40 patients with CHB(60.00%,P = 0.033)and in the 20 HCs(65.00%,P = 0.038).There was no significant difference in the methylation rate of the UBE2Q1 gene in the CHB and HC groups(P=0.707).Fluorescence-based real-time quantitative PCR assays showed that in the pre-ACHBLF group the expression of UBE2Q1 mRNA(7.85 × 10-4 ± 0.96 × 10-4)in patients with UBE1Q1 non-methylation was significantly higher than that in patients with UBE1Q1 gene methylation(3.64 × 10-4 ± 0.55 × 10-4,P<0.001).3.In the pre-ACHBLF patients,no significant differences between the methylation and non-methylation groups in age,gender,level of HBeAg,HBV-DNA viral load,ALT,AST,ALB,or creatinine level(P>0.001).However,among the parameters reflecting the severity of liver failure,which include TBIL,INR,PTA and MELD scores,the TBIL,INR and MELD scores were significantly higher in the non-methylation group than in the methylation group.PTA was significantly lower in the non-methylation group than in the methylation group(P<0.001).Spearman correlation analysis showed that methylation of the UBE2Q1 gene was positively correlated with TBIL and INR levels in patients with pre-ACHBLF(rs = 0.545 and 0.505,respectively;P<0.001),but negatively correlated with PTA level(rs =-0.441,P<0.001).Multivariate logistic regression analysis showed that none of the clinical parameters of patients with pre-ACHBLF were independent prediction factors that reflect UBE2Q1 gene methylation status.4.COX regression univariate analysis suggested that TBIL(P = 0.001),PTA(P =0.036),INR(P = 0.002),MELD score(P<0.001)and hypomethylation status of the UBE2Q1 gene(P<0.001)are early warning factors for progression to ACHBLF from pre-ACHBLF.The above mentioned parameters were included in the multivariate analysis,which showed that the MELD score(P = 0.010)and UBE2Q1 promoter hypomethylation status(P = 0.004)were early warning factors that predict the progression of pre-ACHBLF to ACHBLF.The ROC curve analysis showed that the sensitivity and specificity for early diagnosis of ACHBLF using the methylation status of the UBE2Q1 gene were 92.90%and 65.60%,respectively.The area under the ROC curve(AUC)was 0.792(95%Cl:0.675-0.910).The sensitivity and specificity for early diagnosis of ACHBLF using the MELD score were 96.40%and 62.50%,respectively.The AUC was 0.831(95%CI:0.729-0.934).The ROC indicated that the cut-off point for the MELD score was 19.90.The sensitivity and specificity of UBE2Q1 promoter methylation status combined with the MELD score for early diagnosis of ACHBLF were 92.90%and 75.00%,respectively.The AUC was 0.895(95%Cl:0.818-0.973).ConclusionsHypomethylation of UBE2Q1 gene was associated with severity of Pre-ACHBLF,which was an early warning biological index to judge the development of ACHBLF patients. |
