| Objective Gastric cancer is one of the most common malignancies and the leading cause of cancer-related deaths in the world. Genetic abnormalities of tumor-related genes are well-known to be involved in gastric carcinogenesis. However, another mechanism for inactivation of tumor-related genes is coming more and more into focus. Epigenetic inactivation of certain tumor-related genes by aberrant promoter methylation is frequently observed in gastric carcers and seems to play an important role in both initiation and progression of the carcinogenesis. Methylation profiling of the promoter CpG islands of the known genes has been an important information gathering process for new insights in the new clues for development of the relevant diagnostic and prognostic methods and ever for therapeutics against gastric cancer. Methylation - specific PCR(MSP) was applied to determine the clinicopathological signficance of gene promoter methylation status of 10 genes in 94 cases of gastric cancer. The methylation status of the promoter CpG islands was correlated with sex, age, location, lymph node metastasis, cell differentiation and PTNM phase.Methods Genomic DNA was extracted from tissues using Phenol/Chloroform. DNA samples were treated with bisulfite to convert all unmethylated cytosines to uracil, while leaving methylated cytosines unaffected. Different primers could distinguish methylated/unmethylated status. Then PCR product was sequenced using pGEM-T vector. We also measure the methylation status of adjacent normal tissues treated with Sss I methylase. Results The promoter methylation status of ten genes including E-cad, DAPK, APC, p16INK4a, RASSF1a, hMLH1, TGFβRI, TGFβRII, MGMT and COX-2 were examined. We found that three genes, including E-cad, APC and RASSF1a were methylated in cancer but unmethylated in adjacent normal tissues. Four genes including DAPK, hMLH1, TGFβR II and p16INK4a, were both methylated in cancer and adjacent normal tissues, but the frequencies of methylation in cancer was higher than in adjacent normal tissues. Surprisingly COX-2 was also both methylated in cancer and adjacent normal tissues with higher frequenciey in adjacent normal tissues.In 94cases, the frequencies of methylation for these 10 genes in cancer and adjacent normal tissues were: 34%(32/94) and 0%(0/94)for E-cad, 59%(55 /94) and 3% (3/94) for DAPK,23% (22/94) and 0%(0/94) for APC, 45% (42/94) and 23%(22/94) for p16INK4a, 57%(54/94) and 0%(0/94) for RASSF1a, 28%(26/94) and 5%(5/94) for hMLH1, 12%(11/94) and 8%(7/94) for TGFβRI, 43%(41/94) and 3%(3/94) for TGFβR II, 3%(3/94) and 85%(80/94) for COX-2,16%(15/94) and 26%(24/94) for MGMT respectively.The frequencies of methylation in male and female group were: 32%(19/59) and 37%(13/35)for E-cad, 56%(33/59) and 63%(22/35)for DAPK, 24% (14/59) and 23% (8/35) for APC, 49% (29/59) and 37% (13/35) for p16INK4a, 53%(31/59) and 66%(23/35) for RASSF1a, 31%(18/59) and 23%(8/35) for hMLH1, 12%(7/59) and 11%(4/35) for TGFβRI, 39%(23/59) and 51% (18/35) for TGFβ R II, 15%(9/59) and 17%(6/35) for MGMT respectively.The frequencies of methylation in ≤65 and >65 years group were: 35%(22/63) and 32%(10/31) for E-cad, 52%(33/63) and 71%(22/31)for DAPK, 27%(17/63) and 16%(5/31)for APC, 32%(20/63) and 71%(22/31) for p16INK4a, 49%(31/63) and74%(23/31) for RASSF1a, 24%(15/63) and 35%(11/31) for hMLH1, 14%(9/63) and7%(2/31) for TGFβRI,43%(27/63)and 45%(14/31) for TGFβRII, 19%(12/63) and 10%(3/31) for MGMT respectively. Methylation of pl6INK4a and RASSF1a was more frequent in >65 group. (p<0. 05).The frequencies of methylation in upper, middle and lower part of the stomach groups were:33%(4/12), 35%(9/26) and 34%(19/56) for E-cad, 42%(5/12), 54% (14/26) and 64% (36/56) for DAPK, 33% (4/12) , 23% (6/26) and 21% (12/56) for APC, 58%(7/12), 42%(11/26) and 43%(24/56) for p16INK4a, 67%(8/12), 65% (17/26) and 52% (29/56) for RASSF1a, 33% (4/12) ,27% (7/26) and 27% (15/56) for hMLH1, 17%(2/12), 15%(4/26) and 9%(5/56) for TGFβRI, 50%(6/12),46% (12/26) and 41%(23/56) for TGFβR II, 17%(2/12), 15%(4/26) and 16%(9/56) for MGMT respectively.The frequencies of methylation in no-lymph-node-metastasis and lymph node metastasis group were:35%(9/26) and 34%(23/68) for E-cad, 46%(12/26) and 55%(43/68) for DAPK, 23%(6/26) and 24%(16/68) for APC, 46%(12/26) and 44%(30/68) for pl6INK4a, 46%(12/26) and 62% (42/68) for RASSF1a, 23%(6/26) and 29%(20/68) for hMLH1, 12%(3/26) and 12%(8/68) for TGFβRI , 35%(9/26)and 47%(32/68) for TGFβR II, 15%(4/26) and 16%(11/68)for MGMT respectively.The frequencies of methylation in well, moderate and poor group according to the cell differentiation were:27% (12/45), 39%(14/36) and 46% (6/13) for E-cad, 40% (18/45) ,70% (25/36) and 92% (12/13) for DAPK, 29%(13/ 45), 17%(6/36) and 23%(3/13) for APC, 40%(18/45),56%(20/36) and 31%(4/13) for p16INK4a, 53%(24/45),50%(18/36) and 92%(12/13) for RASSF1a, 22%(10/ 45), 39%(14/36) and 15%(2/13) for hMLH1, 13%(6/45) , 11%(4/36) and 8%(1/13) for TGFβRI, 27%(12/45), 50%(18/36) and 85%(11/13) for TGFβRII, 18%(8/ 45) , 11%(4/36) and 23%(3/13) for MGMT respectively. Methylation of DAPK , TGFβR II and RASSFla were more frequent in moderate and poor group according to the cell differentiation (p<0.05).The frequencies of methylation with PTNM stage I, II, III and IV group were: 26%(4/15),27%(6/22),36%(13/36) and 43%(9/21) for E-cad, 33%(5/15), 23%(5/22), 69% (25/36) and 95% (20/21) for DAPK, 13% (2/15), 36%(8/22), 19% (7/36) and 24%(5/21) for APC, 33%(5/15), 59%(13/22), 39%(14/36) and 48% (10/21) for p16INK4a, 40%(6/15), 41%(9/22), 64%(23/36) and 76%(16/21) for RASSFla,27%(4/15), 32%(7/22), 28%(10/36) and 24%(5/21) for hMLHl, 7%(1/ 15), 9%(2/22), 11%(4/36) and 19%(4/21) for TGFβRI, 27%(4/15), 27%(6/22), 42%(15/36) and 76%(16/21) for TGFβR II, 7%(1/15), 18%(4/22), 17%(6/36) and 19%(4/21) for MGMT respectively . The frequency of methylationfor DAPK , RASSFla and TGFβR II augmented with the PTMN stage. Conclusion Aberrant methylation of the promoter of tumor-related genes is frequently present in gastric cancer. Methylation may be responsable for the decline of transcription of tumor-related genes.Objective To observe the protein expression of the TGF-β/Smad signal system in gastric cancer and discuss the significance in human gastric carcinogenesis.Methods Immunohistochemistry was applied to detect protein expression of TGF-β/Smad signal system in 34 cases of gastric cancer tissues. X~2 test was applied between the frequency for loss of expression. Results The frequency of the loss-or-reduced expression of TGFβRI, TGFβRII and Smad4 was 6% (2/34) , 32% (11/34) 和 12% (4/34) in the gastric cancer tissues respectively. The protein expression of TGFβRII was higher in gastric cancer tissue without hypermethylation. Conclusion The loss of protein expression of TGFβRI, TGF-βRII and Smad4 may lead to the TGF-β resistance and carcinogenesis in gastric cancer .
|
PDF Full Text Request