The Significance Of Methylation Detection For Gene ADAMTS9and RNF180DNA In Plasma With Primary Liver Cancer Patients

Objective: ADAMTS9and RNF180are two newly discovered tumorsuppressor genes, in which ADAMTS9is primarily to inhibit tumor growth byinhibiting angiogenesis, while RNF180plays a role in cell growth anddevelopment process by regulating the ubitination of the target proteinsubstrate. It had been detected that abnormal methylation of these two genes inother tumor tissues in previous. we had also studied the methylation status ofthese two genes in primary liver cancer tissues before, found that the two genemethylation rate in liver cancer and cirrhosis was significantly higher than innormal tissue. It showed that the methylation status of these two genes may beinvolved in the development of liver cancer, and that the methylation status ofthese two genes also had a certain value in liver cancer patient’s condition andprognosis assessment. However, research on tissue is largely traumatic anddifficult to obtain, and it is not suitable for screening and early detection oftumors. In comparison, plasma and serum samples are relatively easy to obtain,and with little traumatic. Previous studies have shown that the DNAmethylation changes in tumor and paired with plasma or serum samples areconsistent, which showed that DNA methylation detection in plasma isexpected to become a new tumor marker. Thus in this study, we detected thepromoter region methylation status of tumor suppressor genes ADAMTS9andRNF180genes in plasma of liver cancer, cirrhosis and healthy controls withmethylation-specific polymerase chain reaction detection method. we alsostudied the methylation status to the diagnostic value of liver cancer, andanalyzed its relationship with clinical data and survival prognosis, filter outnew serological markers which shoud have a significant value for clinicaldiagnosis and prognosis. Methods:1Patients: Plasma samples were collected at the Fourth hospital of HebeiMedical University, Department of Gastroenterology and hepatology,including90cases of liver cancer patients,65cases of cirrhosis and80casesof healthy people,between April2009and September2011. There were no anytreatments before collecting the plasma samples from90cases of liver cancerpatients, including surgery, chemotherapy and interventional treatment. Therewere72males and18females in group of liver cancer, in which the age from33to77and the average age was56.41±10.76. There were45males and20females in group of cirrhosis, in which the age from23to78,and the averageage was58.82±9.85. According to Barcelona clinical liver cancer stage tomake clinical stages for liver cancer, where the cases of stage A, B, C andD were27,16,33and14, respectively. The clinical data and laboratory testresults of all the patients were collected, the survival status of liver cancerpatients were followed up by way of Tel, then calculate the survival time.2Methods of detection for suppressor gene promoter methylation: Thewhole blood (about2ml) which were anticoagulated by EDTA werecentrifuged at2500rpm for2minutes, then the supernatant were obtained asthe resulting plasma samples,DNA from plasma was extracted with QIAampDNA Blood Mini Kit, DNA was methylated and purified with BisulFlashDNA Modification Kit, all steps according to the instructions of the kits, themethylation status of ADAMTS9,RNF180genes in plasma were detected withmethylation specific PCR. The methylation and unmethylation of ADAMTS9gene DNA was amplified with primers,5’-ttgttcgttcgttgggtattatgcg-3’(forward)/5’-ccaacttttaactttaaaaatcgct-3’(reverse),5’-tgtttgtttgttgggtattatgtgg-3’(forward)/5’-ccaacttttaactttaaaaatcact-3’(reverse). The methylation and unmethylation of RNF180geneDNA was amplified with primers,5’-gacgtggggcgagtagggtcgtt-3’(forward)/5’-ccgaccgaaaccaaatccctccgaa-3’(reverse),5’-gatgtggggtgagtagggttgtt-3’(forward)/5’-ccaaccaaaaccaaatccctccaaa-3’(reverse). Amplified the fragment of the tumorsuppressor gene ADAMTS9, RNF180,and the methylation analysis of PCRproducts judged by the results of electrophoresis gel imaging system. 3Statistics analysis: All statistical analyses were performed by SPSS13.0software. P<0.05was considered to indicate a statistically significantdifference.Results:1To compare the methylation status of ADAMTS9,RNF180genes inplasma of liver cancer, cirrhosis and healthy peoplePositive ratio of ADAMTS9gene methylation in plasma of liver cancer,cirrhosis and healthy people were15.56%,3.08%and0%,respectively. Thedifferences among three groups were statistically significant(χ2=18.124,P=0.000<0.05). To compare the methylation status of liver cancerand cirrhosis, there was significantly diffrence between the twogroups(χ2=6.349,P=0.012<0.05). Further more,to compare the healthy controlwith liver cancer, there was significantly diffrence between thegroups(χ2=13.561,P=0.000<0.05). While to compare the healthy control withcirrhosis,there was no significantly difference between thegroups(P=0.199>0.05).Positive ratio of RNF180gene methylation in plasma of liver cancer,cirrhosis and healthy people were6.67%,4.62%and0%,respectively. Thedifferences among three groups were no statisticallysignificant(χ2=5.261,P=0.072>0.05). To compare the methylation status ofRNF180gene in liver cancer and cirrhosis, there was no significantlydifference between the two groups(χ2=0.036，P=0.849>0.05). Further more,tocompare the healthy control with liver cancer and cirrhosis,respectively, therewere no significantly diffrences among the groups(P=0.053，P=0.175).2Comparing ADAMTS9,RNF180gene’s methylation status with theclinical character in plasma of liver cancerThe methylation status of ADAMTS9and RNF180were both not relatedto age, sex, Child pugh score, the value of AFP, number of tumors, with orwithout HBsAg infection,BCLC staging level,with or without cirrhosis, withor without portal vein tumor embolus and with or without transfer,thedifferences between groups were all not statistically significant (P>0.05).Standarded AFP not less than400ug/L as AFP positive, AFP less than400ug/L as AFP negative.The were39cases of AFP positive in90cases ofliver cancer patients, accounting for43.33%. And9cases of ADAMTS9geneand4cases of RNF180gene detecting promoter methylation in the remaining51cases of AFP negative cases, respectively, accounting for17.65%,7.84%.Combined detection of plasma ADAMTS9and RNF180gene methylationpositive rate was18.89%(17/90), combined detection of the methylationstatus of the two genes with AFP detection of liver cancer detection rate was55.56%(50/90).3The diagnostic value by detection of ADAMTS9,RNF180genemethylation in plasmaAmong the235plasma samples of liver cancer, cirrhosis, and healthycontrol, the sensitivity for ADAMTS9gene methylation to predict liver cancerwas15.6%, the specificity was98.6%, repectively. The sensitivity for RNF180gene methylation to predict liver cancer was6.7%, the specificity was97.9%,repectively. The sensitivity of combining the two genes methylation to predictliver cancer was18.9%, the specificity was96.6%, repectively.4The relationship betweenADAMTS9,RNF180gene methylation andsurvival in plasma of liver cancer,and prognostic factors analysis with livercancer patientsThe survival rate of ADAMTS9gene’s methylation status wassignificantly different between the patients with positive(mean survival timewas23.714±6.576months) and negative (mean survival time was16.054±2.028months)(P=0.321>0.05).There was no significant difference in survival rate of RNF180gene’smethylation status between patients with positive(mean survival time was23.000±10.518months) and negative(mean survival time was16.715±2.006months)(P=0.653>0.05).We analysised eight factors of liver cancer patients with Log-Rankunivariate analysis,including age, sex, Child-pugh score, the value of AFP, number of tumors, with or without HBsAg infection, with or without portalvein tumor embolus, BCLC staging level. Found that Child pugh score, withor without portal vein tumor embolus impacted on survival rate of patientswith liver cancer, the difference was statistically significant,(P=0.020, P=0.040).Multivariate analysis showed that Child pugh score,with or without portalvein tumor embolus were independent prognosis factors(P=0.024<0.05，P=0.006<0.05), the other nine factors were not independent prognostic factorsto liver cancer survival prognosis.Conclusions:1Plasma ADAMTS9gene methylation positive rate in plasma of PLCpatients was significantly higher than that of liver cirrhosis group and healthycontrols. It suggests that ADAMTS9gene methylation may play a role in thedevelopment of liver cancer.2The specific degree with detecting methylation status of ADAMTS9and RNF180gene methylation rate in plasma is high for the diagnosis ofPLC,but the sensitivity is low. To combine the two genes methylationdetection and AFP detection, the sensitivity for liver cancer diagnosis is notenhanced very well,which indicate that this indicators is not suitable as amarker for diagnosing of liver cancer, and also not suitable for screeninghigh-risk populations.3The methylation status of plasma suppressor gene ADAMTS9andRNF180can not be an indicators for evaluation and prognosis of patients withliver cancer.Child-pugh classification and presence of portal vein tumorthrombus can be evaluated as an independent prognostic factor of liver cancersurvival prognosis.