The Study On PAI-1Genetic Instability,Methylation In5’-flanking Region,BTG3Promoter Methylation And The Relationship With Clinicopathological Features In Chinese Gastric Cancer

Part Ⅰ Study on Genetic Instability and CpG Methylation in5’-flanking Region of the PAI-1Gene in Chinese with Gastric CancerBACKGROUND:Gastric cancer remains one of the commonly seen tumors in clinical practice in a Chinese population with the highest mortality. Therefore, great attention should be paid to finding new early markers and effective treatment methods for this disease.Carcinogenesis is a multistep process that results from the accumulation of the alterations. Genetic instability of oncogenes, such as microsatellite instability (MSI) and loss of heterozygosity (LOH), is probably associated with mutations of genes responsible for tumor-genesis, which play an important role in tumor pathology. On the other hand, epigenetic regulation of gene expression seems to play a major role in cancer. The silencing of tumor suppressor genes by aberrant hypermethylation in human cancer have been documented.Plasminogen activator inhibitor-1(PAI-1), regulates the activities of the tissue-type plasminogen activator (tPA) and the urokinase-type plasminogen activator (uPA). Plasmin-catalyzed degradation of basement membranes is believed to enable cancer cells to invade normal tissue. The tumors contain higher amounts of the PAI-1than the corresponding normal tissue. Therefore, a high level of PAI-1in tumors is a good marker of poor prognosis.OBJECTIVE:This research was to explore the correlation of genetic instability and CpG methylation in the5’-flanking region of the PAI-1gene to clinicopathologic features of gastric cancer in Chinese patients, and provide experimental basis for revealing the molecular markers for diagnosing gastric tumor’s development.METHODS:Microsatellite instability (MSI) and loss of heterozygosity (LOH) of locus D7S515, D7S471and pai-1in50specimens of gastric cancer and relevant pericancerous tissues were detected by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) with ordinary sliver staining. Methylation-specific PCR (MS-PCR) was used to analyze the CpG methylation in the5’-flanking region of the PAI-1gene. SPSS statistical software was used to analyze the relationship between experimental results.RESULTS:The detection rates of MSI was higher in negative than in the positive serosa infiltration group of gastric cancer (42.86%vs.2.33%, P=0.031). The frequency of MSI was significantly lower in the cases with lymph node metastasis than in those without metastasis (18.18%vs.2.56%, P=0.007), whereas it was higher in the low differentiation group than that in middle or high differentiation groups (21.05%vs.0.00%, P=0.027). The CpG methylation in the5’-flanking region of the PAI-1gene did not show significant difference.CONCLUSIONS:MSI and LOH of PAI-1gene regulate the development of gastric cancer through different pathways. The occurrence of MSI may be a molecular marker for the development of gastric cancer. The CpG methylation in the5’-flanking region of the PAI-1gene may not take part in the canceration of gastric tissues. Part II Genistein Upregulate Expression of BTG3By Reversing Hypermethylation and Induces G2/M Cell Cycle Arrest in Gastric CancerBACKGROUND:Epigenetic alterations are heritable changes in gene expression without an accompanying change in primary DNA sequence. Histone modification and DNA methylation, two major epigenetic processes, can control the expression of genes at the transcriptional level. There is increasing evidence that aberrant DNA methylation and histone acetylation are useful independent molecular prognostic markers. For example, hypermethylation of CpG islands in promoter regions of the gene occur earlier than malignant proliferation. Thus, cancer could be diagnosed at an early stage of development. The epigenetic regulation of gene expression plays a major role in cancer. Aberrant transcriptional silencing of tumor suppressor genes by epigenetic deregulation is a common occurrence in human cancers.B-cell translocation gene3(BTG3/ANA/APRO4) is a candidate tumor suppressor gene in some malignancies. We report here that BTG3is transcriptionally down-regulated in gastric cancer. BTG3is thought to be a negative regulator of the cell cycle. More recently, researchers found that BTG3promoter hypermethylation has been associate with human non-small cell lung cancer breast cancer, oral squamous cell carcinomas, renal cancer and so on. But there is no research about those relationship in gastric cancer.Since epigenetic changes are reversible, inhibitors of DNA methylation could raise the possibility of exploiting new therapeutic targets. Genistein had similar effects to that of5Aza, which is a potent demethylating agent with high toxicity and instability. On the other hand, Genistein being a natural, non-toxic, dietary isoflavone is effective in retarding the growth of prostate cancer cells, and already have the clinical research. It is a promising candidate for epigenetic therapy in human cancer.OBJECTIVE:1、We detect the expression of BTG3in gastric cancer. And research the relationship between gene expression and promoter hypermethylation.2、 Genistein had similar effects to that of5Aza. It is a promising candidate for epigenetic therapy in human cancer.3、Genistein induces G2/M cell cycle arrest in gastric Cancer.METHODS:BTG3mRNA expression was determined by quantitative real-time polymerase chain reaction; BTG3protein expression was determined by Western blot. Bisulfite-modified polymerase chain reaction, cloning and sequencing were used to examine promoter methylation; Gastric cancer cell line7901、AGS、803、K45and normal gastric cell line Ges-1were treated with genistein and5-aza-2’-deoxycytidine. Fluorescence-activated cell sorting was performed to analyze cell cycle and apoptosis, and cell proliferation was examined by cell viability assay(CCK-8).RESULTS:We found that BTG3mRNA and protein expression was down-regulated in cancer tissues and cell. Genistein and5Aza-C induced BTG3mRNA and protein expression in gastric cancer cell line, but not in normal gastric cell line Ges-1. Furthermore, genistein and5Aza-C treatment significantly decreased promoter methylation in a putative methylation target regions in gastric cancer cell line, reactivating BTG3expression. Genistein has antiproliferative effect on cancer cell growth through induction of G2-M cell cycle arrest.CONCLUSIONS:These results suggest that silencing of BTG3expression through hypermethylation of the promoter leads to loss of its tumor-suppressive activity, which may be a factor in the carcinogenesis of gastric cancer. Genistein showed similar effects to that of5Aza-C, and our results indicate that genistein is a novel, advantageous therapeutic agent for treating gastric cancer.