| BackgroundAngioimmunoblastic T-cell lymphoma(AITL)is a kind of Peripheral T-cell lymphoma(PTCL).The etiology and pathogenesis of the disease is still not clear,and there is no unified treatment program.The median survival time after traditional chemotherapy is usually less than 3 years.The formation and development of AITL may be controlled by genetic mechanism and epigenetic mechanism,which is a cascade process of multi-gene particpation.Gene NR4A3 belongs to the NR4A family and participates in cell cycle,apoptosis,inflammation,metabolism and so on.We analyzed the methylation rate and degree ofNR4A3 methylation in AITL.The aim of this study was to identify biomarkers for their early diagnosis.Methods(1)A total of 60 paraffin embeded tissue of angioimmunoblastic T-cell lymphoma and 50 reactive hyporplasia as control group during 2005.01-2014.11 were collected.(2)DNA were extracted from sliced paraffin embeded tissue.(3)5 pairs of The experimental group and the control group were selected to analyze the difference of methylation status between them by Illumina methylation gene chip and statistical methods.(4)Several tumor suppressor genes were selected based on results of illumina methylation gene chip and methylation specific PCR was performed in a large sample cases to obtain a gene with significant difference in methylation rate between the experimental group and the control group.(5).The degree of promoter methylation of 53 experimental and 29 control samples was quantitatively analyzed by pyrosequencing.(6)BSP was performed to test the methylation status of Jurkat cell line.(7)Clinical data such as patient sex,age,tumor size,ki67 and LDH were collected,Follow up were conducted.Kaplan-meier method and log-rank test were used to plot the survival curve and statistically analyze the survivability of the tumor.Results(1)Methylation gene chip shows that abnormal DNA methylation is a very common event in the globle genome of AITL tumor tissue.Abnormal methylation outside the gene body is mostly happened in the promoter region.Abnormal methylation is more common in CpG shore region.KEGG and GO analysis were enriched to gain the relevant signaling pathways and biological functions.Hypermethylation status of tumor suppressor genes CCDC67,KCNH2,BCL2L10,RNF41,BBC3,P21,sept9,NEIL1 and NR4A3 were showed by results of the methylation gene chip of experimental versus control samples.(2)The methylation rate of gene was analyzed by methylation-specific PCR.The results showed that the methylation rate of NR4A3 in angioimmunoblastic T cell lymphoma was higher than control group,the difference was statistically significant(P<0.05).While no significant difference of the rate of methylation in other tumor suppressor genes between two group were found.(3)The results of sequencing ofpyrophosphate showed that NR4A3 had a higher degree of methylation in angioimmunoblastic T cell lymphoma than control group(P<0.05)(4)The hypermethylation methylation status of NR4A3 in Jurkat lymphoma cell line was confirmed by BSP.(5)Age,sex,tumor size,Ki67,LDH and other factors were analysed by Kaplan-meier method and log-rank test,LDH was significantly correlated with overall survival(OS)(P<0.05).ConclusionAbnormal DN A methylation is a very common event in the globle genome of AITL tumor tissue.Abnormal methylation of the region other than the gene body occurs most frequently in the promoter region and the 5’UTR region,the CpG shore region and the island.5’UTR of NR4A3 was hypermethylated in AITL.The hypermethylation status of can surve as a new biomarker for diagnosis.Patients with higher LDH tend to have a poor prognostic of AITL. |
PDF Full Text Request