Diagnostic And Prognostic Significance Of CCND1 And Ran Promoter Hypomethylation In Primary Hepatocellular Carcinoma

Part Ⅰ Hypoethylation of CCND1 Promoter in Hepatitis B Virus-Associated Hepatocellular CarcinomaBackgroundPrimary hepatocellular carcinoma(HCC)is a high-incidence malignancy of digestive disease,which is in the forefront of fatal diseases.This disease is the main histological type of primary liver cancer,with dormant onset and difficulty to detect in the early stage.Primary liver cancer was the sixth most largest cancer and the third leading cause of cancer death across the world,with about 906000 new cases and 830000 deaths in 2020.The prognosis of patients with primary liver cancer is poor,and the 5-year survival rate is merely 18%.Most patients with HCC have the background of hepatitis B virus(HBV)infection.Patients with chronic liver diseases have persistent liver inflammation,fibrosis and abnormal hepatocyte regeneration,which lead to cirrhosis and s series of epigenetic events,and finally the formation of premalignant lesions(dysplastic nodules).DNA methylation refers to that a specific base on the DNA sequence using a S adenosyl methionine(SAM)as a methyl doner under the catalysis of DNA methyltransferase(DNMT)to form 5-methylcytosine(5-mC)by covalent binding.The changes of gene promoter methylation can cause oncogene activation,tumor suppressor gene inactivation and abnormal expression,which promotes the occurrence and development of tumor.In recent years,with the deepening of the research on liver cancer,researchers found that the aberrant of DNA methylation is closely related to its development.This type of epigenetic alteration is biologically stable and exists in circulating tumor DNA(ctDNA),which makes it suitable for early detection and noninvasive dynamic monitoring of tumor.Professor Motokura T and others of Harvard University School of Medicine found a candidate oncogene PRAD1 on chromosome 11q13.They later discovered that the complementary DNA of PRAD1 encodes a protein composed of 295 amino acids,and its sequence is similar to cyclin.Now the gene is called CCND1,and its encoded protein is Cyclin D1,a regulatory subunit of cell cycle kinase(CDK)4 and 6,whose main function is to promote cell proliferation.Overexpression of CCND1 alters the process of cell cycle and may lead to tumorigenesis.However,the role of CCND1 promoter methylation and its mRNA expression in the diagnosis and prognosis of HBV-HCC needs to be further clarified.Reactive oxygen species(ROS)is highly active substances containing oxygen free radicals.The imbalance between the production of ROS and antioxidant defense may cause oxidative stress,which subsequently cause cell dysfunction and tissue damage.Excessive oxidative stress is widely considered to be a key factor in tumorigenesis.Studies have shown that DNA damage caused by oxidative stress leads to a large number of DNA hypomethylation and contributes to the progression of HCC.However,the relationship between CCND1 promoter methylation and oxidative stress is not clear.Objectives1.To explore CCND1 promoter methylation status in patients with HBV-HCC,patients with chronic hepatitis B(CHB)and healthy controls(HCs).2.To investigate the diagnostic and prognostic values of CCND1 promoter methylation in patients with HBV-HCC,AFP negative HBV-HCC and AFP positive CHB.MethodsOne hundred and five patients with HBV-HCC and 54 patients with CHB were randomly included in our study,all of whom were inpatients or outpatients in the Department of Hepatology,Qilu Hospital of Shandong University from May 2016 to July 2018.Additionally,32 healthy volunteers were included as normal controls.Methylation specific polymerase chain reaction(MSP)was used to detect the CCND1 promoter methylation status of plasma DNA in all patients and HCs.The expression levels of CCND1 mRNA in peripheral blood mononuclear cells(PBMCs)of all patients and HCs were detected by quantitative real-time polymerase chain reaction(RT-qPCR).Enzyme linked immunosorbent assays(ELISAs)were used to detect the levels of superoxide dismutase(SOD),8-hydroxydeoxyguanosine(8-OHdG)and malondialdehyde(MDA)in plasma.Results1.The CCND1 promoter methylation frequency in plasma DNA of patients with HBV-HCC was significantly lower than that of patients with CHB and HCs(x2=25.520,P<0.001;χ2=31.219,P<0.001).2.The CCND1 promoter methylation status was negatively correlated with the plasma levels of SOD,8-OHdG and MDA(rs=-0.533,P<0.001;rs=-0.324,P=0.001;rs=-0.526,P<0.001).3.The area under the receiver operating characteristic(AUROC)of CCND1 promoter methylation in the diagnosis of HBV-HCC was significantly higher than that of AFP(0.705 vs 0.531,P<0.05).The value of combined detection of CCND1 promoter methylation and alpha fetoprotein(AFP)in the diagnosis of HBV-HCC was significantly higher than that of CCND1 promoter methylation or AFP alone.4.In the diagnosis of patients with AFP-negative HBV-HCC,the AUROC of CCND1 promoter methylation was significantly higher than that of AFP(0.717 vs 0.559,P<0.05).In the diagnosis of patients with AFP-positive CHB,the AUROC of CCND1 promoter methylation was significantly higher than that of AFP(0.881 vs 0.800,P<0.05).5.The expression level of CCND1 mRNA in PBMCs of patients with HBV-HCC was significantly higher than that of CHB patients and HCs(Z=-4.946,P<0.001;Z=-6.819,P<0.001).In patients with HBV-HCC,the expression level of CCND1 mRNA in CCND1 promoter methylated group was significantly lower than that in CCND1 promoter unmethylated group(Z=-2.268,P=0.023).The CCND1 promoter methylation status was negatively correlated with its mRNA expression level(Z=-0.213,P=0.032).6.Compared with patients with CHB and HCs,the levels of plasma oxidative damage and antioxidant damage indexes(MDA,SOD and 8-OHdG)in patients with HBV-HCC were significantly increased(P<0.05).In patients with HBV-HCC,the levels of oxidative damage and antioxidant damage indexes(MDA,SOD and 8-OHdG)in CCND1 promoter methylated group were higher than those in CCND1 promoter unmethylated group(P<0.05).7.The overall survival(OS)of CCND1 promoter methylated group was significantly higher than that of unmethylated group in patients with HBV-HCC(P=0.012,log-rank test).Univariate and multivariate Cox regression analysis showed that plasma 8-OHdG level,CCND1 promoter methylation and TNM stage were independent risk factors influencing the prognosis of patients with HBV-HCC.8.In patients with HBV-HCC,the progression free survival(PFS)in the CCND1 promoter methylated group was higher than that in the unmethylated group(HR=0.109,95%CI:0.031-0.384).ConclusionsPatients with HBV-HCC showed a significantly lower CCND1 promoter methylation frequency than patients with CHB and HCs.There was no obvirous difference in the CCND1 promoter methylation frequency between patients with CHB and HCs.the CCND1 mRNA expression level in HBV-HCC patiens with CCND1 promoter unmethylated group was higher than that of CCND1 methylated group.The levels of plasma MDA,SOD and 8-OHdG in patients with HBV-HCC were higher than those in patients with CHB and HCs.The levels of plasma MDA,SOD and 8-OHdG in HBV-HCC patients with methylated CCND1 promoter were reduced than those with unmethylated CCND1 promoter.The diagnostic potential of CCND1 promoter methylation outperformed serum AFP in patients with HBV-HCC.The promoter methylation status of CCND1 gene in plasma DNA served as an independent risk factor influencing the prognosis of patients with HBV-HCC.The OS of HBV-HCC patients with CCND1 promoter methylated was higher than those with CCND1 promoter unmethylated.The methylation status of CCND1 promoter was related to oxidative stress,and plays a crucial biological role in the tumorigenesis,and possesses favorable diagnostic and prognostic significance for HBV-HCC.Part II Expression Level and Promter Methylation Level of Ran and Prognostic Value in Hepatocellular CarcinomaBackgroundHepatocellular carcinoma(HCC)serves as the main histological type of liver cancers,and 83.9%-92.3%of liver cancers are HCC in China.HCC has a high degree of malignancy,and the vast majority of diagnosed patients are in the middle or advanced stage.As a country with high incidence of liver cancer,China accounts for more than 50%of new liver cancer cases and deaths worldwide.According to statistics,in 2014,there were 364800 new cases and 318800 deaths of liver cancer in China.Although the incidence rate of age standardized HCC has decreased in recent years,it is still one of the high risk factors affecting national health,and has become a major problem that in the medical field.Serum alpha fetoprotein(AFP)has been widely used in the screening,diagnosis,evaluation of treatment effect and prediction of recurrence of HCC.However,abnormal AFP mainly occurs in the late stage of the disease.Some studies have pointed out that the false positive rate and false negative rate of serum AFP in screening HCC are relatively high,and there are limitations in distinguishing HCC from benign liver diseases.The serum AFP level of a considerable number of HCC patients might not increase,and the AFP level of benign liver diseases such as viral hepatitis could also increase.In different cohort studies,the areas under the receiver operating characteristic curve(AUROC)of AFP were between 0.73 and 0.83.The study of biomarkers of HCC is of great significance for early diagnosis and prognosis.There is an urgent need to find effective biomarkers to guide treatment and evaluate prognosis.In addition to extensive genetic changes,the occurrence and development of malignant tumors are also closely related to epigenetics.Epigenetic changes exist in all human tumors.At present,it is known that it contributes cancer progression and development together with genetic changes.Epigenetic abnormalities are common in all stages of cancer development,so they can be used to early detection and predict prognosis.DNA methylation is the most deeply studied epigenetic modification.DNA demethylation may lead to genomic instability and aneuploidy,both of which are typical features of cancer.In addition,DNA hypomethylation leads to increased chromosome fragility.Compared with somatic mutation detection,DNA methylation analysis has some unique advantages in tumor recognition,such as higher clinical sensitivity,larger dynamic range,more methylation targets and multiple changeable CpG sites in each target gene region.Ras related nuclear protein(Ran),a small GTP binding protein,is a munber of Ras superfamily.It is involved in regulating the transport of macromolecules through the nuclear membrane,and is also related to mitotic spindle assembly,cell cycle progression and nuclear envelope formation.Like other GTPases,Ran depends on the conformational changes of GTP and GDP binding,interacts with effector proteins and regulates the above biological processes.Ran overexpression has been reported in a variety of tumor types,including renal,breast,ovarian,lung and colon cancer and malignant pleural mesothelioma.However,the expression patterns of Ran in HCC and normal liver tissues are not clear,and the value of Ran mRNA expression level and its methylation level in the diagnosis and prognosis of HCC still needs to be further clarified.Objectives1.To analyze the mRNA expression levels and promoter methylation levels of Ran in HCC and normal liver tissues.2.To study the diagnostic and prognostic value of Ran mRNA expression level and methylation level in HCC patients.3.To investigate the relationships between Ran mRNA expression level and immune cell infiltrations.MethodsIn The Cancer Genome Atlas(TCGA)database,we analyze the correlation between Ran mRNA expression level and its promoter methylation level,and explore the prognostic value of Ran mRNA expression level and its promoter methylation level in HCC patients.The correlations between Ran mRNA expression level and immune cell infiltrations were studied in Tumor Immune Estimation Resource(TIMER)database.Sixty-eighty HCC patients hospitalized in Qilu Hospital of Shandong University from December 2019 to November 2020 were selected to verify the Ran mRNA expression levels in HCC cancer tissues and normal liver tissues by real-time quantitative polymerase chain reaction(qRT-PCR).Results1.In TCGA-LIHC data set,the expression levels of Ran in cancer tissues were higher than that in normal liver tissues(P<0.05).RT-qPCR analysis showed that the expression levels of Ran in cancer tissues was significantly higher than that in adjacent tissues(P=0.002).2.The expression level of Ran mRNA was an independent risk factor affecting the prognosis of patients(HR=1.943,95%CI:1.388-2.720).3.The areas under the time-dependent receiver operating characteristic curve of Ran mRNA expression in predicting 1-year,3-year and 5-year mortality in patients with HCC were 0.747,0.634 and 0.704,respectively.4.Kaplan-Meier analysis showed that the overall survival(OS)and progression-free survival(PFS)of high Ran expression group were significantly lower than those of low Ran expression group(P<0.001).5.Spearman rank correlation analysis showed that the promoter methylation level of Ran was negatively correlated with its expression level(r=-0.36,P<0.001),and methylation levels of CpG sites cg01060409,cg17629322,cg25201101,cg01758167 and cg19847963 were significantly negatively correlated with Ran mRNA expression level(r=-0.26,P<0.001;r=-0.36,P<0.001;r=-0.33,P<0.001;r=-0.16,P<0.005;r=-0.23,P<0.001).6.The OS of cg17629322 and cg25201101 hypermethylated groups were higher than that of hypomethylated groups(P<0.05),whereas the PFS of cg01060409 and cg19847963 hypermethylated group were significantly higher than that of hypomethylation group(P<0.05).7.Compared with pathological grade 3 and 4 HCC patients,pathological grade 1 HCC patients had lower Ran mRNA expression levels(P<0.001,P<0.05).The Ran mRNA expression levels of HCC patients with TNM stage I were significantly lower than that of HCC patients with TNM stage Ⅱ and Ⅲ(P<0.05,P<0.001).The Ran mRNA expression levels of HCC patients with T1 stage were significantly lower than that of HCC patients with T2 stage and T3 stage(P<0.05,P<0.001).The Ran promoter methylation levels of HCC patients aged≥65 years were higher than that of HCC patients aged<65 years(P=0.041).The Ran promoter methylation levels of HCC patients with TNM stage Ⅰ were significantly lower than that of HCC patients with TNM stage Ⅱ and Ⅳ(P=0.03,P=0.026).The Ran promoter methylation levels of HCC patients with T1 stage were higher than that of HCC patients with T2 and T4 stage(P=0.049,P=0.048).8.The Ran mRNA expression levels of HCC patients was positively correlated with the infiltration levels of B cells,CD4+T cells,myeloid dendritic cells,macrophages and neutrophils(P<0.05),and was correlated with prognosis related immune genes.ConclusionThe Ran mRNA expression levels in HCC tissues were significantly higher than that in normal liver tissues,but negatively correlated with the Ran promoter methylation levels.The expression level of Ran mRNA and its promoter methylation level were related to the clinicopathological features of HCC,which may affect the prognosis of HCC.