Razi Journal of Medical Sciences

fa پروفایل متیلاسیون نواحی پروموتری MINT1،MINT2 ، MINT25 و MINT31 در بیماران مبتلا به سرطان معده Methylation Profile of MINT2, MINT1, MINT25 and MINT31 Promoter Regions in Patients with Gastric Cancer ژنتیک Genetic پژوهشي Research <div style="text-align: justify;">زمینه و هدف: ویژگی CpG island methylator phenotype (CIMP) که در بدخیمی‌های متعدد مشاهده می‌شود، یک مجموعه تغییرات اپی ژنتیکی است که با متیلاسیون پروموتر و خاموشی سرکوبگرهای تومور مرتبط است. پژوهش حاضر به بررسی پروفایل متیلاسیون نواحی پروموتریMINT1،MINT2 ، MINT25 وMINT31 در بیماران مبتلا به سرطان معده پرداخته است. روش کار: در این مطالعه بیماران سرطان معده از نوع آدنوکارسینوما در سنین بین 50 تا 85 سال مشارکت داشته و نمونه برداری از بافت‌های سرطانی و کنترل با روش آندوسکوپی انجام شد. طبق پروتکل کیت‌های انتخابی، DNA بافت‌ها استخراج و سپس با بیسولفیت تیمار گردید. با استفاده از روش MS-PCR نواحی مورد نظر تکثیر و بر روی ژل پلی آکریل آمید ارزیابی شدند. یافته‌ها: نتایج به دست آمده نشان داد که 9/52% از بافت‌های آدنوکارسینومای معده  CIMPمثبت بوده که شامل 2/41% CIMP-Low و 7/11% CIMP-High می‌شدند. مارکرMINT25 با 76% مثبت، بیشترین میزان متیلاسیون را به خود اختصاص داده بود. این در حالی است که در گروه کنترل هیچ مورد مثبت MINT25 مشاهده نشد که می‌تواند تاییدی بر اختصاصیت این مارکر باشد. در رتبه بعدی MINT31  با 53% مثبت قرار داشت. ژن‌های MINT1 و MINT2 به ترتیب در 7/17 درصد و 8/11 درصد موارد مثبت ارزیابی شدند. ارتباط آماری معنی‌داری بین مصرف داروهای مهارکننده‌ی پمپ پروتون و متیلاسیون نواحی پروموتری MINT25 و MINT31 به دست آمد. نتیجه‌گیری: فنوتیپ CIMP قابلیت معرفی به عنوان یک بیومارکر در تشخیص سرطان معده را دارد که البته انتخاب پروفایل متیلاسیون باید بر اساس ویژگی‌های هر جمعیت استاندارد سازی شود. نتایج مطالعه حاضر نشان داد که از بین 4 ناحیه ژنی پیشنهادی MINT25 ارتباط آماری معنی داری با سرطان معده و همچنین با سابقه مصرف داروهای مهارکننده‌ی پمپ پروتون دارد که دال بر اهمیت بررسی این مارکر در بیماران ایرانی مبتلا به سرطان معده است.</div> <div style="text-align: justify;">Background & Aims: Gastric cancer is the fifth most common cancer and the fourth leading cause of cancer death in the world. This genetically heterogeneous condition can be sporadic or inherited as an autosomal trait. One of the most important causes of sporadic gastric cancer is environmental factors such as Helicobacter pylori infection. In recent decades, proton-pump inhibitors (PPIs) have been widely used to treat gastrointestinal problems. New researches raise concerns about the link between PPIs and gastric cancer. The CpG island methylator phenotype (CIMP) seen in various malignancies is a set of epigenetic changes associated with promoter methylation and down regulation of the tumor suppressor genes. The present study investigated the methylation profile of MINT2, MINT1, MINT25 and MINT31 promoter regions in patients with gastric cancer. The MINT1 gene, also known as Amyloid-beta A4 precursor protein-binding family A member 1 (APBA1), is located on 9q and encodes a protein which is involved in signal transduction processes. Studies have shown a significant increase in MINT1 hypermethylation in gastric cancer tissues significantly associated with Helicobacter pylori infection. The MINT2 gene, or Amyloid-beta A4 precursor protein-binding, family A, member 2 (APBA2), has been mapped to the 15q region. Abnormal methylation of MINT2 has been identified in the blood and peritoneal fluid of patients with gastric cancer suggesting that it can be used as a predictor of peritoneal micrometastasis. The next two gene regions, MINT25 and MINT31, are functionally different. In fact, MINT25 is the Matrix metalloproteinase 24 (MMP24) gene, located at 20q. Studies have shown that MINT25 hypermethylation is a specific molecular marker for gastric cancer screening. The MINT31 marker is located at 17q in the non-coding region upstream of the CACNA1G gene, which is a T-Type Calcium Channel. MINT31 plays an important role in regulating CACNA1G expression. Studies have shown that hypermethylation of the MINT31 region can be used as a predictor of gastric cancer progression. Based on these findings, the present study investigates the methylation profile of MINT2, MINT1, MINT25 and MINT31 promoter regions in patients with gastric cancer. Another goal of this project was to investigate the correlation between this methylation profile and patients' clinical characteristics. Methods: In this descriptive-analytical study, patients with adenocarcinoma gastric cancer between the ages of 50 and 85 years old were studied. The age matched control group included non-cancerous gastrointestinal patients who referred for endoscopy. Tumor and control tissue samples were collected by endoscopy. Collected tissue samples were extracted using the GENTBIO kit and DNA samples were kept at -20. Methylation-specific PCR (MSP) was used to determine the methylation pattern of selected gene regions. Bisulfite treatment was performed by Bisulfite Conversion Kit (ZYMO RESEARCH). Briefly, bisulfite solutions were prepared according to the kit instructions and were added to the samples. The mixtures were incubated for 5 hours then centrifugation was performed. In the next step a combination of BL buffer and carrier RNA were added to the products. The samples were then thoroughly mixed with ethanol and transferred to Epitect columns. BW wash buffer and BD desulfurizing buffer were used in later stages to improve DNA quality. For each reaction of PCR we used 2X PCR-master mix, 0.5µl of each forward and reverse primers with a concentration of 10μM and 50ng DNA. The following temperature program was applied: 15 minutes at 95°C, 35 cycles including 20 seconds at 95 °C, 30 seconds annealing with variable and specific temperature for each pair of primers, extension for 25 seconds at 72°C and final extension for 5 minutes at 72°C. The annealing temperature of the primer pairs was standardized between 51-60°C. PCR products were evaluated by 8% polyacrylamide gel stained with silver nitrate. After collecting and extracting the results, the data were analyzed using SPSS software version 19. P value less than 0.05 was considered significant. Results: Methylation profile of MINT2, MINT1, MINT25 and MINT31 promoter regions was assessed on polyacrylamide gel after treatment with bisulfite and MS-PCR. Each gene was amplified by a pair of primers for methylated (M) and a pair of primers for non-methylated (U) regions. In case of positive methylation of one or two genes, the situation was considered CIMP-Low phenotype and in case of positive methylation of 3 or 4 genes, phenotype was recorded as a CIMP-High. If the methylation of all four genes was negative, the CIMP-Negative phenotype was recorded. The results showed that CIMP was positive in 52.9% of gastric adenocarcinoma tissues, which included 41.2% CIMP-Low and 11.7% CIMP-High. MINT25 with 76% positive had the highest methylation rate. Interestingly, no positive cases of MINT25 were observed in the control group which could confirm the specificity of this marker. MINT31 was next with 53% positive cases. In control samples for three promoter regions, methylation results were negative and only two positive cases were observed regarding MINT31. MINT1 and MINT2 genes were evaluated positive in 17.7% and 11.8% of cases, respectively. According to these results, there was a statistically significant correlation between PPIs and methylation of MINT25 and MINT31 promoter regions. Considering all 4 gene regions as CIMP phenotype, the significance of the results was reduced, which could be due to the low sample size of positive cases in MINT1 and MINT2. Contrary to expectations, no significant link was observed between the Helicobacter pylori infection and methylation of each gene alone. However, the CIMP-Positive phenotype showed a stronger association with Helicobacter pylori infection than individual genes, although this association was not reached the significant level. Conclusion: Gastric cancer is a multifactorial disease that despite the discovery of several predisposing genes, scientific evidence still indicates the important role of environmental factors in its development. Studies have shown the effective role of epigenetic changes in gastric carcinogenesis. Methylation of many genes has been studied in gastrointestinal cancers, including gastric cancer. Epigenetic instability, characterized by hypermethylation of multiple CpG islands, is known as the CIMP phenotype. CIMP-positive tumors indicate that hypermethylation has occurred simultaneously in the promoter regions of several tumor suppressor genes. This leads to inactivation of gene transcription and consequent loss of their function. So far, different gene sets have been selected to study CIMP in a variety of cancers, due to the heterogeneity of histological features in tumors and patient populations. In the present study, the promoter regions of MINT1, MINT2, MINT25 and MINT31 in patients with gastric cancer were examined and the results confirmed the presence of CIMP-Low and CIMP-High phenotypes in these samples. In our study, the correlation between methylation pattern and clinical characteristics of patients was also investigated. Statistical analyzes showed a significant link between methylation of MINT25 and MINT31 promoter regions with the PPIs. Evidence from several studies suggests that long-term use of PPIs is associated with a higher risk of gastric cancer. However, some studies have suggested that this risk may be limited to people with a history of Helicobacter pylori infection or precancerous lesions of the stomach. Because PPIs induce hypergastrinemia and cellular hyperplasia, they could increase the risk of gastric cancer. Overall, the results of our study show that the CIMP phenotype can be introduced as a biomarker in the diagnosis of gastric cancer, although the methylation profile selection should be standardized based on the characteristics of each population. The results of our study showed that among the 4 proposed gene regions, MINT25 has a statistically significant correlation with gastric cancer and also with a history of PPIs, which indicates the importance of studying this marker in Iranian patients with gastric cancer. </div> سرطان معده, متیلاسیون DNA, جزایر CpG, مهارکننده‌‌های پمپ پروتون Gastric cancer, DNA Methylation, CpG islands, Proton Pump Inhibitors 95 105 http://rjms.iums.ac.ir/browse.php?a_code=A-10-6481-1&slc_lang=fa&sid=1 Nasibe Tabibi Jebeli نسیبه طبیبی جبلی 3900319475328460074859 000000034404004X No Department of Medical Genetics, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran گروه ژنتیک پزشکی، دانشکده علوم پزشکی، دانشگاه تربیت مدرس، تهران، ایران Shirin Shahbazi شیرین شهبازی sh.shahbazi@modares.ac.ir 3900319475328460074860 0000000276345350 Yes Department of Medical Genetics, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran گروه ژنتیک پزشکی، دانشکده علوم پزشکی، دانشگاه تربیت مدرس، تهران، ایران Elham Tabesh الهام تابش 3900319475328460074861 0000000185337945 No Department of Internal Medicine, Faculty of Medical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran گروه داخلی، دانشکده پزشکی، دانشگاه علوم پزشکی اصفهان، اصفهان، ایران