Volume 30, Issue 1 (3-2023)
RJMS 2023, 30(1): 95-105 |
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Research code: IR.MODARES.REC.1397.145
Ethics code: IR.MODARES.REC.1397.145
Clinical trials code: 0
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Tabibi Jebeli N, Shahbazi S, Tabesh E. Methylation Profile of MINT2, MINT1, MINT25 and MINT31 Promoter Regions in Patients with Gastric Cancer. RJMS 2023; 30 (1) :95-105
URL: http://rjms.iums.ac.ir/article-1-7277-en.html
URL: http://rjms.iums.ac.ir/article-1-7277-en.html
Department of Medical Genetics, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran , sh.shahbazi@modares.ac.ir
Abstract: (768 Views)
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.
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.
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