Mechanistic And Prognostic Significance Of Aberrant Methylation Of ADAMTS9in Human Hepatocellular Carcinoma

Objective: Hepatocellular carcinoma(HCC) is one of the most frequentlydiagnosed tumour diseases throughout the world, with increasing incidenceand mortality. However, the molecular mechanisms of hepatocarcinogenesisare far from clear. With the development of epigenetics,more and moreattentions focused on the role of the methylation of tumor suppressor genes intumorigeness and development. The human ADAMTS (a disintegrin andmetalloproteinase with thrombospondin-like motifs) family was involved in awide range of biological processes includingECM assembly and degradation,hemostasis, organogenesis and the regulation of angiogenesis. ADAMTS9is amember of ADAMTS family and it is a newly discovered tumor suppressorgene. According to the previous report, promoter hypermethylation of theADAMTS9gene existed in esophageal cancer, nasopharyngeal carcinoma,gastric cancer, colon cancer and pancreatic cancer. But the mechanistic andprognostic significance of aberrant methylation of ADAMTS9in humanhepatocellular carcinoma are far from clear. In this research, hepatoma cells,fresh liver tissue and paraffin-embedded tissue specimens were used to detectADAMTS9gene methylation level and gene protein expression. To invistigatethe relationship between the gene and the clinical data, pathologicalcharacteristics and lifetime of the data analysis of the gene in hepatocellularcarcinomathe significance of the progress and prognosis,in order to find themechanistic and prognostic significance of aberrant methylation ofADAMTS9in human hepatocellular carcinoma. To provide theoreticalsupport for the tumor suppressor gene ADAMTS9DNA promoter methylationin clinical detection.Methods:1Cell culture and treatment: Hepatoma cell line Hep-G2routinely cultured in RPMI-1640culture medium, SMMC-7721hepatoma cell lineroutinely cultured in DMEM medium. Two cell lines were cultured in theculture medium which contained5-aza-2’deoxycytidine (5-Aza-CdR) finalconcentration of10umol/L for24h, after that replaced the free5-Aza-CdRculture medium and then incubated for72h. Extract DNA in both case anddetect the methylation status of ADAMTS9gene with methylation specificPCR（MSP） method were done.2Subjects and specimens:132patients with hepatocellular carcinomawere enrolled in our study who were taken surgical resection at the FourthHospital of Hebei Medical University Hepatobiliary Surgery form April2009to January2011.113cases were male and19females, aged23to78years andthe average age of54.29±9.78years. Tumor diameter range from0.5cm to25cm.123cases of liver function Child A, B+C grade nine cases. Clinicalstage of the132cases were different, there were40cases with stage Ⅰ,andstage Ⅱ76cases, stage Ⅲ15cases and stage Ⅳ1case. All patientsbefore and after surgery were not received any adjuvant therapy. Thefollow-up to collect patient survival data.All specimens were279cases, including fresh132cases ofhepatocellular carcinoma tissue specimens and corresponding adjacent132cases of cirrhosis tissues of15cases of normal liver tissue as a control.Specimens embedded in paraffin HE staining, two pathologist for histologicaldiagnosis. Well differentiated in25cases, and midle differentiation of the21cases,86cases of poorly differentiated and undifferentiated.3ADAMTS9methylation status detection: Methylation-specific PCR(Methylation Specific PCR, MSP) was used to detect ADAMTS9methylationstatus in132cases of hepatocellular carcinoma and corresponding cirrhosistissues and15cases of normal tissue.4ADAMTS9gene protein detection: detected76cases of hepatocellularcarcinoma tissue and corresponding cirrhosis tissue and10patients withnormal liver tissue ADAMTS9protein expression with SP method.5Using the SPSS13.0for statistical analysis. Measurement data using t-test and non-parametric tests,the count data using the X~2test or Fisher’s exacttest,survival analysis using the Kaplan-Meier method, univariate analysisusing the Log-rank test, multivariate analysis using COX model. P <0.05considered statistically significant.Results:1The ADAMTS9gene DNA methylation status in Cancer cells andtumor tissue.There are three conditions of ADAMTS9gene DNA methylation status.Fully methylated state, hemimethylated state and completely unmethylatedstate.2Before and after the treatment of liver cancer cell growth stateobservation and ADAMTS9of gene methylation status detection2.1Cell growth state observerBefore treated with5-Aza-CdR Hep-G2, SMMC-7721was the state ofnormal growth. After treated with5-Aza-CdR,the two cell lines presentedapoptosis.2.2Detection of ADAMTS9gene methylation in both cell linesThe methylation status of ADAMTS9in Hep-G2，SMMC-7721werepositive,and the status of Hep-G2is fully methylated and SMMC-7721Hemi-Methylated.After treated with5-Aza-CdR The methylation status of ADAMTS9inHep-G2，SMMC-7721were both negative.And completely unmethylated bandexisted.3ADAMTS9gene methylation status in hepatocellular carcinomaspecimens and the relationship between the clinical and pathological data.3.1Comparing the methylation of genes ADAMTS9in cancerous,adjacent tissues and the normal liver tissue.Positive ratio of ADAMTS9gene DNA promotor methylation incancerous tissues and adjacent tissues and normal tissues were66.67%(88/132),29.55%(39/132),0.0%(0/15). Significant difference betweenthree groups was exist.Cancer tissue methylation positive rate was significantly higher than the adjacent tissues and the normal tissues.3.2Comparison of the ADAMTS9gene methylation status in patientswith different clinical and pathological featuresThe ADAMTS9gene DNA promotor methylation frequency in patientspositive rate referring to gender(p=0.220),age(p=0.175),childclassification(p=0.717), AFP(p=0.428), tumor size(p=0.452), tumornumber(p=0.869),HBsAg status (p=0.632),tumor metastasisstatus(p=0.497),pathology differentiation level(p=0.875)were not statisticallysignificant (P>0.05). Portal vein tumor thrombus in patients withmethylation-positive rate (78.72%) greater than patients with portal veintumor thrombus methylation-positive rate (60.00%), the difference wasstatistically significant (p=0.029). The positive rate of ADAMTS9DNAmethylation in the patients with stage I to II was greater than the positive ratein the patients with stage Ⅲ to Ⅳ(p=0.008).4ADAMTS9gene protein expression in hepatocellular carcinomaspecimens and the relationship between the clinical and pathological data.4.1The protein expression of ADAMTS9in hepatocellular carcinomaspecimens25cases of the hepatocellular carcinoma ADATMS9gene proteinexpression was positive, the positive rate of32.89%,76cases of cirrhosistissue54cases ADATMS9gene protein expression, the positive expressionrate was71.05%,10cases normal liver tissue, the nine cases ADATMS9geneprotein expression, the positive expression rate was90%.The differencesbetween the three groups was statistically significant (p=0.000), the positiverate of cancer tissue positive rate (p=0.001) lower than the cirrhosis tissue (p=0.000) and normal tissue(p=0.001).4.2Comparison of the ADAMTS9gene protein expression in patientswith different clinical and pathological features.The protein expression of ADAMTS9referring to gender,age,childclassification,the value of serum AFP,tumor size,portal vein tumor embolusstatus,tumor number,HBsAg status were not statistically significant (P>0.05). Significant differences of ADAMTS9gene protein expression between thetumor metastasis status (p=0.041),pathology differentiation level(p=0.003)and clinical stage(p=0.046).5Relationship between methylation status of ADAMTS9and itsexpression of protein.The protein expression of ADAMTS9in methylated tumortissues(17.07%) was significantly lower than that in unmethylated tumortissues(48.64%)(P=0.036).6Relationship between the promoter region of the tumor suppressor geneADAMTS9DNA methylation status and the survival time.Kaplan-Meier analysis of ADAMTS9methylation positive patientsshowing two-year survival rate was29.5%, the median survival time of10months, the average survival time was12.50±0.93months; negative patients,two-year survival rate was50.0%,the median survival time of23months, theaverage survival time was19.61±1.19months, the difference was statisticallysignificant (p=0.002). The metastasis patients with two-year survival rate was23.8%, no metastasis patients with two-year survival rate was42.2%, and thedifference was statistically significant (p=0.036). In order to analyze theindependent factors relating to the total survival time of hepatocellularcarcinoma patients,12variables were selected to conduct the COXAnalysis.The variables are gender, age, Child level, AFP value, tumor size,portal vein tumor embolus status, tumor number, HBsAg status, tumormetastasis, differentiated level, clinical period and methylation status. Theanalysis demonstrated that methylation of ADAMTS9is an independentprognostic factor of hepatocellular carcinoma.Conclusions:1Positive ratio of ADAMTS9gene DNA promotor methylation innormal tissues, adjacent tissues and cancerous tissues in turn increased,indicate that abnormal methylation of the promoter region of the ADAMTS9may be involved in the formation of hepatocellular carcinoma.2There is a relationship between methylation status of ADAMTS9gene and with or without portal vein tumor thrombus formation and clinical stage,indicating that ADAMTS9methylation status reflects the hepatocellularcarcinoma patients in a certain extent.3The methylation status of ADAMTS9is related to the prognosis ofhepatocellular carcinoma, and is an independent prognostic factor ofhepatocellular carcinoma.