Research On Abnormal Methylation Of Several Tumor-suppressing Genes In Human Gastric Cancer

The activation of oncogenes or inactivation of tumor suppressor genes is one of the key steps in the molecular processe of tumorigenesis. Transcriptional silencing of many Tumro - supressing Gene for promoter hypermethylation of tumor suppressor genes is a well — recognized mechanism. In contrast to the genetic alterations, DNA methylation is classified as the epigenetic alterations which can be replicated stably and inherited during cell proliferation. The aberrant pattern of methylation could be reversed more easily, and such reverse process, especially in the early stage of tumorigenesis, is important in the treatment of tumors. Theoretically, re - expression of tumor suppressor genes by demethylating agents may contribute to inhibit the development of tumor. It has been suggested that DNA hypermethylation results in resistance to chemotherapy and consequently reduces the efficacy of chemotherapeutic treatment. Therefore, utilization of demethylating agents synergistically in enhancing the sensitivity of chemotherapy becomes a new and hot spot.It has been reported that the DNA methylation alters the expressions of certain genes. Methylation - silenced tumor suppressor genes are detected in some cancers. It is found that we can re - express some silenced tumor suppressor genes containing CpG Island via demethylating pathway and reverse their tumor suppressing function in vitro. These observations provide a novel therapeutic strategy in anticancer trestment. However, there is few systematic study on DNA methylation in gastric normal tissue, primary and metastatic tumor, and few reports of the effects of dementhylating agents on the chemotherapy and cell growth of gastric cancer have been seen.We want to study tumor suppressor genes methylation in gastric normal tis-sue, primary and metastatic tumor and the relationship between tumor suppressor genes methylation and clinical pathological characteristics to find the molecular mechanism of gastric cancer carcinogenesis and a new way of gene therapy.Materials and MethodsMaterials: 78 surgical specimens of gastric carcinoma resceted at the First Affiliated Hospital of China Medical University from March, 1998 to May, 2005 were studied including 54 males and 24 females ( average age is 56. 2 ± 7. 5 years. ) , 20 cases of Primary gastric carcinoma and 58 cases of progressive gastric carcinoma, 44 cases of differentiated type and 14 cases of undifferentiated type. 10 mmol/L hydroquinone and 3. 9 mol/L sodium bisulfite obtained from Sigma Company;DNA Clean - up System from Promega Company;TIMP3 antigen and SP kit from Dalian Bostor Biotechnology Company.MethodsImmunohistochemistry;Immunohistochemistry was used to detect the protein expression of TIMP3^THBS1 NMINT1 AE - cadherin and P16 in the tissues of gastric cancer.MSP;Methylation Sensitive Polymerase Chain Reaction was used to detect tumor - suppressing gene methylation in normal tissue, primary and metastatic tumor respectively.RT - PCR: Using RT - PCR to compare mRNA expression level of these genes in gastric cell lines treated with 5 - aza - dC with those in gastric cell lines before being treated.Western blot: Using Western blot to compare protein expression level of these genes in gastric cell lines treated with 5 - aza - dC with those in gastric cell lines before being treated.ResultsMSP detection : we detect promoter of tumor - suppressing gene in normal gastric tissue ,primary tumor, progressive tumor and metastatic lymph node respectively in 78 cases and find the positive rate of P16 is 10. 3 % ,35.0 % ,41. 4 % and 65.2 %;THBS1 is 6.4 % ,40. 0 % ,48. 4 % and 52. 2 %;TIMP3 is14.1 %,50.0 % ,51. 7 % and 58. 7 %;MINT1 is 11.4 %,45.0 %,41.3 % and 60. 9 %;E - cadherin is 12. 8 % ,65. 0 % ,72. 4 % and 78. 3 %. The difference among normal tissue, primary and metastatic tumor is significant in statistic test (P <0.05). so the promoter methylation of PI6 NTHBSUTIMP3, MINT1 and E - cadherin may be involved in the carcinogenesis and metastasis of gastric cancer.Immunohistochemistry: we detect protein expression of several tumor — suppressing genes in normal gastric tissue , primary tumor, progressive tumor and metastatic lymph node respectively in 78 cases and find the positive rate of P16 is 97.4 % ,30.0 % ,27.6 % and 17.9 %;THBS1 is 89.7 % ,40.0 % ,31. 0 % and 14.1 %;TIMP3 is 94.9 % ,45.0 % ,36.2 % and 15.4 %;MINT1 is96.2 % ,50.0 % ,41.4 % and 20.5 %;E -cadherin is 100 % ,35 % ,37.9 % and 20. 5 %. Either The difference between normal tissue and primary tumor or normal tissue and metastatic tumor is significant in statistic test (P <0.05). So the expression of TIMPS^THBSl ^MINTl ,E - cadherin and P16 decrease in primary and metastatic tumor. The protein expression has inverse correlation with the methylation level.RT - PCR: Compare the mRNA levels of tumor - suppressing gene in four human gastric cell lines with those in the four human gastric cell lines treated with methylation inhibitor we find that the mRNA levels is increased in the latter . Intensity of every strap deepen in every concentration in 4 human gastric cell lines human gastric cell lines methylation inhibitor 5 - aza - dC . The average ratio of tumor - supressing gene expression strap density value to the (3 - actin in cell lines treated with methylation inhibitor is higher than that in those not treated. The difference is significant in statistic test (P <0.05). No mRNA differ-ence is detected in every concentration in the cell lines treated with methylation inhibitor.Western Blot: Compare the protein expression levels of tumor - suppressing gene in four human gastric cell lines with those in the four human gastric cell lines treated with methylation inhibitor we find that the protein expression is increased in the latter. Intensity of every strap deepen in every concentration in 4 human gastric cell lines human gastric cell lines methylation inhibitor 5 - aza -dC . The average ratio of tumor - supressing gene expression strap density value to the (3 - actin in cell lines treated with methylation inhibitor is higher than that in those not treated. The difference is significant in statistic test (P < 0. 05 ). No protein expression difference is detected in every concentration in the cell lines treated with methylation inhibitor.ConclusionThe expression of tumor - suppressing gene TIMP3NTHBS1AE - cadherin^ MINT1 and PI 6 expression decrease in gastric cancer tissue than in normal tissue . Difference of tumor - suppressing gene expression in gastric cancer tissues with different cell differentiation, metastasis lymphonode and infiltration depth is significant. Tumor - suppressing gene expression level is an important index for tumor malignanacy and prognosis.There is tumor - suppressing gene methylation in gastric cancer and metastasis lymphonode. It has relationship with different stage of gastric cancer.Expressions of TIMPS^THBSl^nmlS^E - cadherin and PI 6 are decreased in gastric cell lines. Promoter region hyper - methylation is probably the main mechanism. Demethylating agent 5 - aza - dC can re - express them.Demethylating agent can increase protein expression of P16>,E - cadherin N THBSKTIMP3 and nm23 in gastric cancer line SGC -790UBGC -823NMKN -45 and MGC -803 in vitro. Promoter methylation is probably one of the main reasons.Demethylating agent 5 - aza - dC can re - express tumor - suppressing gene in gastric cancer and this function has hereditability.Tumor - suppressing gene methylation is a potential indicator for gastric cancer molecular diagnosis and evaluation.