Studies On The Methylation Detection Of Genes Associated With Gastric Cancer And Demethylation With Combination Of Drugs

Gastric cancer is one of the most common malignancies and the leading cause of cancer-related deaths in the world , which is still a menace to human health. Surgery is still the best choice, but the five-year survival is less than 30%. Although people have made obvious progress in surgery, radiotherapy, chemiotherapy, the prognosis of patients is still disturbing. Among the reasons of the high mortality of gastric cancer, lack of specific early diagnosis marker and effective treatment is the most important reason, but little is known about the mechanisms of gastric cancer initiation, development and progression by far. People once only focused on DNA sequence changes in some tumor associated genes such as amplification of oncogenes , mutation and inactivation of tumor suppressor genes, but recently it was found that the hypermethylated status of promoter CpG islands is correlated with the expression silencing of the tumor suppressor genes and implicated as the 2nd hit. While the reverse process, loss of methylation can also recruit in the activation of the otherwise inert genes including some oncogenes. Epigenetics is described as a heritable change in gene expression without any changes in DNA sequence.Therapy of Gastric cancer is still a problem for surgeons, so, it's more important to study gastric cancer in molecular level, to learn more about the molecular events and the corresponding mechanisms in gastric cancer carcinogenesis. DNA methylation plays a key role in gene expression, regulation, cell division and differentiation, development and carcinogenesis. If the methylations occurs in regulators, such as promoter, enhancer, inhibitor et, those kinds of changes will suppress the gene's functions. Epigenetics studies have revealed that promoter methylation isone of the mechanisms of tumor suppressor gene (TSG) inactivation. According to Knudson's "two hit theory", the alleles of TSG can be inactivated by methylation, or with the help of mutation, deletion. When tumor cell lines with promoter hypermethylation were treated with demethylation drugs, such as 5-Aza or isomer, the TSG could re-express the mRNA and proteins, and the re-expression level would change corresponding with different cell lines and different amount of drugs. So, those tests proved that promoter methylation is one of the main mechanisms of gene expression inactivation. Combined with function studies of TSG and the mechanism of methylation-related gene inactivation, those indicated that the methylation of CpG islands could directly cause malignancy. Aberrant DNA methylation locus has become the most potential molecular marker in early detection, risk evaluations, prediction of response to chemotherapy and prognosis of cancer patients.Recent studies have focused on some TSG genes such as PI6, ECD ,hMLHl and so on which can suppress the malignancy of tumor. Aberrant promoter methylations of these genes were found in many tumors, and the expressions were often lost in tumor cell lines. But how about the expression level of PI6, ECD ,hMLHl in gastric cancer , is the promoter methylated? If so, is promoter methylation the main mechanism for the loss of gene expression? Is demethylation drug effective to the target? Can combination of demethylation drug Aza and SPB reverse the process or recruit in the activation of the genes in gastric cancer cell line or nude mice with implanted gastric cancer cell line. We began the study of therapy,transcriptional expression and promoter hypermethylation of PI6, ECD and hMLHl in clinical gastric cancer samples, gastric cancer cell line and nude mice with implanted gastric cancer..PURPOSE: To discover the molecular changes and mechanism in gastric cancer initiation and progression process and find some effective molecular marker in epigenetic level for gastric cancer diagnosis and treatment in early stage by studing protein expression and CpG islands methylation status of PI 6 -. hMLHl > E-CD gene in gastric cancer samples.MATERIALS AND METHODS:1.Forty-one gastric carcinomas , 40 premalignant lesions and 38 normal control samples were collected randomly . The specimens were divided into two groups and reserved in -80 °C. One group was used to detect 5' methylated cytosine phosphate guanosine in gene promoter region by MSP;another group was used to detect PI6 > hMLHl > E-CD expression by immunohistochemistry.2.Human gastric carcinoma cell line SGC-7901 was treated by methylation inhibitor, 5-aza-2' — deoxycytidine(5-Aza) and sodium phenylbutyrate (SPB). The methylation, transcription and expression level of E-CD, PI6 and hMLHl gene in different test groups were analyzed by MSP, RT-PCR, Western blot, immunofluorescence and immunocytochemistry.3.The cell inhibitory index was determined by the change of cell morphology under light microscope and MTT study.4. Cell cycle and apoptosis of SGC-7901 cells after treated were detected with Flowcytometry (FCM).5. The apoptosis of gastric cancer cell lines SGC-7901 was also evaluated by HE staining, electron microscope and DNA ladder test.6. SGC-7901 cells were transfected into BABL/c nude mouse.7 Nude mice transfected with gastric carcinoma cell line SGC-7901 were treated by methylation inhibitor, Aza and SPB. The methylation, transcription and expression level of E-CD, PI6 and hMLHl gene in different test group were analyzed by MSP, RT-PCR, Western blot, and immunocytochemistry.8. Observe the tumor body change and paint the tumor growth curve.9 .The apoptosis of tumor cells in nude mice was analyzed with HE staining, electron microscope, and DNA ladder test.RESULTS:1. In 41 gastric cancer samples, the frequency of P16> E-CD and hMLHl expression is51.22%(21/41) , 70.73%(29/41), 60.97%(25/41) respectively, which is higher than that in premalignant lesions and normal control group. P value is less than 0.05.2. In 41 gastric cancer samples, the frequency of CpG islands methylation status of PI 6 , hMLHl , E-CD gene is 56.09% (23/41) , 19.51% (8/41), 34.15% (14/41) respectively, which is lower than that in premalignant lesions and normal control group. P value is less than 0.05.3. The data showed that PI6 , hMLHl, E-CD gene can be demethylated by 5-Aza-CdR and SPB which can significantly elevate the transcription and expression of P16 , hMLHl, E-CD at both mRNA and protein leves in SGC7901 cell lines. The results showed that the demethylated E-CD, PI6 and hMLHl gene by Aza and SPB might significantly inhibit cell proliferation, migration, enhance cell adhesion, and induce cell apoptosis, as well as, make morphological changes in vitro.4. MTT results showed that Aza and SPB can inhibit the growth rate of gastric cancer SGC7901 cells in vitro. The inhibitory rates of gastric cancer SGC7901 cells treated with SPB, Aza, Aza and SPB group were 54.6%,67.5% and 78.7% respectively.5. The data of FCM indicated that the growth of SGC-7901 cells was directly suppressed in treated groups: S phase and G2-M phase cells reduced dramatically and G0/G1 phase cells increased significantly. PI (proliferative index) was 51.53%, 48.68%, 35.68% and 21.76% in different test groups respectively, which decreased significantly in treated groups compared with control group.6.HE staining, electron microscope and DNA ladder test indicated that the apoptosis cells could be detected when treated with Aza and SPB. Under the electron microscope, the significant structure of apoptosis was showed.7.The nude mouse model of gastric cancer cell line SGC-7901 was constructed successfully.8. The weight and volume of tumor body reduced after being treated with Aza and SPB. The volume of tumor body in 4 test groups was 2.26cm3,1.30cm3,1.17cm3 and 0.63cm3 respectively, (PO.05). And inhibitory rates of tumor volume was 42.4%, 48.23%, 72.12% in 3 treated groups respectively.9 The results of HE staining and electron microscope indicated that Aza and SPB could induce apoptosis in gastric cancer of nude mouse. Cell apoptosis rate increased from 4.28% to 5.12%, 14.63% and 23.22% (PO.05). Under the electron microscope, the significant structure of apoptosis was showned.10 The data showed that P16 > hMLHK E-CD gene can be demethylated by 5-Aza-CdR and SPB which can significantly elevate the transcription and expression of PI6 , hMLHK E-CD at both mRNA and protein leves in implantation tumor of nude mice. Mice gastric cancer model injected with Aza and SPB showed a significant inhibition of the metastasis of tumor with lighter lung weight, a fewer metastasis nodules with respect to the control group. The results of experiments show restored E-CD, P16 and hMLHl gene has a significant antitumor effect.CONCLUSION:1. P16 -. hMLHl .. E-CD methylation may be early events in gastric carcinogenesis and can be good parameters for early diagnosis, predict biological character and prognosis.2. Demethylation treated with Aza and SPB can restore the gene expression of PI 6 % hMLHl-. E-CD, inhibit cell proliferation, migration, enhance cell adhesion, and induce cell apoptosis in SGC7901 cell line whatever in vitro or vivo. AzaR and SPB had a significant antitumor effect, which were responsible for demethylation.3. Aza and SPB are promising new therapeutic medicines and demethylation reagents for antitumor Gene methylation could become a target for tumor therapy, which provide an innovative approach to cancer therapy. Our experimental result will supply valuable data and clue for clinical apply of Aza and SPB.