.28 A Tumor-associated Gene Promoter Cpg Island Methylation And Transcription Associated With Non-small Cell Lung Cancer Study

Objective Lung cancer is one of the most common malignancies and theleading cause of cancer-related deaths in the world. Non-small cell lungcancer(NSCLC) account for 70-80% of lung cancer. The most effectivetreatment for lung cancer is surgical resection.Many patients undergoingsurgical resection will ultimately die of recurrence of NSCLC,suggestingthe frequent presence of occult metastatic lession.Genetic abnormalitiesof oncogenes and tumor suppressor genes(TSGs) are well-known to beinvolved in lung cancer pathogenesis.However,another mechanism forinactivation of TSGs is coming more and more into focus.Epigeneticinactivation of certain TSGs by aberrant promoter methylation isfrequently observed in lung carcinomas and seems to play an important rolein both initiation and progression of the carcinogenesis.Methylationprofiling of the promoter CpG islands of the known genes has been animportant information gathering process for new insights in the new cluesfor development of the relevant diagnostic and prognostic methods andever for therapeutics against lung cancer. To determine theclinicopathological signficance of gene promoter methylation status of28 genes in 10 free-cancer pulmonary diseases and 43 NSCLCs by methylation–specific PCR(MSP). The methylation status of the promoter CpG islandswas correlated with sex,age,smoking ,pathological type,pathologicalphase and TNM phase.The relationship between CpG methylation and mRNAtranscription in four lung cancer cell lines was also observed..Methods Genomic DNA was extracted from tissues using Phenol /CHCl2.DNAsamples were treated with bisulfite to convert all unmethylated cytosinesto uracil,while leaving methylated cytosines unaffected.Differentprimers could distinguish methylated/un-methylated status.Then PCRproduct was sequenced using pGEM-T vector.We also measure the methylationstatus of benigh pulmonary tissues treated with Sss I methylase.Thesemi-quantitative RT-PCR method was used to assess the expression of sixgenes in three lung cancer cell lines.Results The promoter methylation status of twenty-eight genes includingAPC,AR,ABO,DAPK,GSTPI,MAGEA1,MGMT,MYOD1,p16INK4a,p21WAF,p14ARF,p15INK4b,p73,RASSF1a,WT1,Rb,TIMP-3,VHL,OCT6,N33,E-cad,CDH13,DNMT1,PTEN,hMLH 4复旦大学 2004 届博士研究生论文1,RAR-β,mint31 and cox-2 were examined.We found that 12 genes were bothunmethylated in NSCLCs and benigh pulmonary diseases group.Two genes,MAGEand N33,were both methylated in NSCLCs and benigh pulmonary diseases.Fourteen genes,including E-cad,DAPK,APC,MYOD1,p16INK4a,WT1,CDH13,MGMT,RASSF1a,RAR-β,p73,AR,p21WAF and OCT6 were methylated in NSCLCsbut unmethylated in benigh pulmonary diseases.In 43 NSCLC cases,thefrequencies of methylation for these 14 genes in NSCLCs and correspondingnon-neoplastic lung tissues were: 23% and 19% for E-cad, 35% and 26% forDAPK, 21% and 23% for APC, 40% and 19% for MYOD1, 42% and 44% for p16INK4a,51% and 28% for WT1, 37% and 19% for CDH13, 26% and 26% for MGMT,35% and21% for RASSF1a,28% and 19% for RAR-β,44% and 16% for p73, 23% and 16%for AR, 28% and 16% for p21WAF,23% and 9% for OCT6 ,respectively. Thefrequencies of methylation in male and female group were:22% and 27% forE-cad,34% and 36% for DAPK,22% and 18% for APC,41% and 36% for MYOD1,34% and 64% for p16INK4a,50% and 55% for WT1,41% and 27% for CDH13,28%and 18% for MGMT, 34% and 36% for RASSF1a, 25% and 36% for RAR-β,44% and45% for p73,19% and 36% for AR,28% and 27% for p21WAF,22% and 27% forOCT6,respectively. The frequencies of methylation in ≤65 and >65 yearsgroup were: 24% and 22% for E-cad, 32% and 44% for DAPK, 21% and 22% forAPC, 41% and 33% MYOD1, 47% and 22% for p16INK4a, 50% and 56% for WT1,35% and 44% for CDH13, 29% and 11% for MGMT,26% and 67% for RASSF1a,26% and 33% for RAR-β,41% and 56% for p73, 26% and 11% for AR, 32% and11% for p21WAF, 26% and 11% for OCT6.Methylation of RASSF1a was morefreq...