Optimizing a methylation-specific polymerase chain reaction technology / 中国职业医学

OBJECTIVE: To establish a highly specific methylation-specific polymerase chain reaction( MSP) method.METHODS: Bisulphited conversion hypermethylated human genomic DNA and unmethylated human genomic DNA were used as template and p15 gene as the target gene. The Cp G island of p15 gene promoter was predicted by Methprimer software.The methylated and unmethylated primers for polymerase chain reaction( PCR) of methylated and unmethylated DNA templates were designed. The PCR stage of MSP was divided into normal temperature group( methylation primer and unmethylated primer annealing temperature of 58 and 55 ℃,respectively) and elevated temperature group( methylation primer and unmethylated primer annealing temperature were 60 and 57 ℃ respectively). The PCR reaction system was optimized based on the normal temperature group by increasing the concentration of magnesium ions( Mg2 +) and adding dimethyl sulfoxide( DMSO) in the PCR reaction system. RESULTS: Nonspecific amplification products appeared in normal temperature group in the PCR reaction stage of MSP,indicating false positive and false negative reactions. The non-specific amplification was weakened and the specific amplification was equally reduced in the elevated temperature group. The nonspecific amplification disappeared in the optimized PCR system and normal temperature group,and the false positive and false negative reactions were eliminated completely. CONCLUSION: Setting the normal annealing temperature and the concentration of Mg2 +and DMSO is beneficial to ensure the specificity of MSP results.

Relationship between methylation status of Rap1 GTPase activating protein and colon cancer / 肿瘤研究与临床

Objective To explore the methylation status of Rap1 GTPase activating protein (Rap1GAP) promoter in colon cancer, and to provide the oretical basis and research direction for the early diagnosis, targeted therapy, anti-multidrug resistance of colon cancer and so on. Methods The paraffin embedded specimens of 33 patients with colonic adenocarcinoma diagnosed by pathology were analyzed from Department of Pathology of Xinzhou City People′s Hospital from January 2010 to September 2014, including 19 males and 14 females, and aged 41-72 years old. The paraffin embedded specimens of 16 patients with colonic adenoma were enrolled, including 9 males and 7 females, and aged 34-58 years old. 13 normal tissues from the tumor distal margin (from the tumor > 15 cm) were selected. Quantitative methylation specific PCR (q-MSP) was applied to detect methylation level of Rap1GAP gene promoter. The methylation level differences of Rap1GAP gene promoter region among 3 groups or between different clinicopathologic factor subgroups were compared. Results The methylation rates [median (interquartile range)] of Rap1GAP promoter were 65.43 % (50.35 %), 21.37 % (8.39 %) and 17.43 % (15.71 %) in colonic adenocarcinoma group, colonic adenoma group and adjacent normal tissue group, respectively. The methylation rate of colonic adenocarcinoma group was significantly higher than that of colon adenoma group or that of adjacent normal tissue group (P60yearsold:36.26%(62.62%)and26.23%(76.42 %);well-differentiated vs. moderately/poorly-differentiated: 21.98 % (40.32 %) vs. 42.74 % (74.20 %); TNM Ⅰ-Ⅱ vsⅢ-Ⅳ: 25.31 % (48.27 %) vs. 36.26 % (75.55 %); all P> 0.05]. Conclusion The methylation status of RAP1GAP promoter maybe associate with genesis and development of colon cancer, which might be used as a target for early diagnose of colon cancer.

IGF-II gene promoter P3 methylation in salivary pleomorphic adenoma / 实用口腔医学杂志

Objective:To detect the methylation status of insulin-like growth factor Ⅱ(IGF-Ⅱ)gene promoter P3 in salivary pleo-morphic adenoma(SPA).Methods:The methylation of IGF-Ⅱ gene promtor P3 was examined in 26 cases of salivary pleomorphic adenoma with adjacent normal salivary gland tissues,1 0 cases of salivary gland malignant tumor (except malignant pleomorphic ade-noma)and 1 0 cases of normal salivary gland tissue by nested methylation specific polymerase chainreaction(nMSP),1 0 samples(3 SPA and controls,2 malignance and controls)were examined by pyrosequencing.Results:9 out off the 26 SPA showed lower level methylation oevel of IGF-II gene promoter P3.Normal salivary gland and salivary gland malignant tumor tissue did not.Conclusion:IGF-II gene promoter P3 with low level of methylation may play a role in the development of SPA.

Isolation of human genomic DNA from archival dried blood spots for neonatal disease screening and its application to methylation detection / 中华实用儿科临床杂志

Objective To establish an effective DNA isolation method for neonatal disease screening,so as to explore its application to the methylation detection.Methods The 20 dried blood spots samples were randomly divided into 2 groups according to the gender:the traditional method group (n =10) and the improved kit method group(n =10).The DNA quality was evaluated based on its concentration,integrity and whether it could be used in polymerase chain reaction (PCR).These DNA samples with or without bisulfite treatment were used as template in the methylation-specific polymerase chain reaction (MSP).The methylation levels of Leptin and tumor necrosis factor-α (TNF-α) gene promoter region were detected.Results DNA concentration of the improved kit method [(5.70 ± 0.81) mg/L] was significantly higher than that of the traditional method [(3.50 ± 0.45) mg/L] (t =2.79,P ＜ 0.05),and biochemical analyzer analysis showed a better DNA integrity.Agarose gel electrophoresis revealed that 18S gene fragment could be successfully amplified by PCR method,suggesting its potential application to PCR study.MSP results showed different DNA methylation levels of Leptin and TNF-α genes promoter regions from various samples.Conclusions The improved kit method can effectively extract DNA from dried blood spots samples,and these DNA can be used in methylation research.The study can provide a new research direction and technical method to reveal the pathogenesis of disease from the perspective of DNA methylation.

Diagnostic value of promoter methylation and protein expression of plasma RNF180 gene in gastric cancer / 中国肿瘤临床

Objective:To investigate the diagnostic value of the promoter methylation of plasma RNF180 gene and its protein ex-pression for the detection of gastric cancer. Methods:Methylation-specific polymerase-chain reaction (MSP) and enzyme-linked immu-no-sorbent assay (ELISA) were performed to detect DNA methylation and protein expression of the RNF180 gene, respectively. The correlations of DNA methylation and protein expression of the RNF180 gene with the clinico-pathological parameters of gastric carcino-ma were then separately analyzed. Results:MSP showed that the methylation rates of the RNF180 gene were 62.75%and 21.88%in the plasma of patients with gastric carcinoma and healthy volunteers, respectively;this result indicated that the two groups significantly differed (P0.05). Conclusion:The RNF180 gene is expressed at a hypermethylation rate, and the corresponding protein expression level is de-creased in the plasma of individuals with gastric carcinoma. Therefore, RNF180 gene methylation in plasma could be applied to detect microinvasion for the clinical diagnosis of gastric cancer.

Methylation status of CDH13 gene promoter in colon cancer / 中国医师进修杂志

Objective To detect the methylation status of CDH1 3 gene promoter in colon cancer by using methylation-specific polymerase chain reaction(MSP)technique.Method The tissue specimens from 32 cases of colon cancer(observation group),and 12 cases of normal colon tissues(control group)were examined,the methylation of CDH13 gene promoter was detected by MSP.Result The methylation of CDH13 gene promoter was detected in 19 cases(59.4％,19/32)in observation group,1 case(8.3 ％,1 / 12)in control group,there was statistical significance between two groups(P =0.002).Conclusion Frequency of the methylation of CDH13 gene promoter is apparently higher in colon cancer tissues than that in normal colon tissues,it reveals that CDH13 gene promoter may contribute significantly to the development of colon cancer.

A Consideration of MGMT Gene Promotor Methylation Analysis for Glioblastoma Using Methylation-Specific Polymerase Chain Reaction and Pyrosequencing

BACKGROUND: O6-methylguanine-DNA methyltransferase (MGMT) gene promoter methylation is currently the most promising predictive marker for the outcome and benefit from temozolomide treatment in patients with glioblastoma, but there is no consensus on the analysis method for assessing the methylation status in the molecular diagnostic field. The objective of this study was to evaluate methylation-specific polymerase chain reaction (MSP) and pyrosequencing methods for assessing MGMT gene promotor methylation of glioblastoma as well as assessing the MGMT protein expression by immunohistochemistry. METHODS: Twenty-seven cases of glioblastoma from the archives at the Department of Pathology Konkuk University Hospital were selected. MGMT promoter methylation was evaluated by MSP and the pyrosequencing methods. The MGMT expression was also measured at the protein level by immunohistochemistry. RESULTS: Overall, MGMT hypermethylation was observed in 44.4% (12/27 cases) of the case of glioblastoma using either MSP or pyrosequencing. The concordant rate was 70.3% (19/27 cases) between MSP and pyrosequencing for MGMT methylation. There was no correlation between MGMT methylation and the protein expression. No significant differences in progression free survival and overall survival were seen between the methylated group and the unmethylated group by using either MSP or pyrosequencing. The status of the MGMT protein expression was correlated with progression free survival (p=0.026). CONCLUSIONS: In this study the concordance rate between MSP and the pyrosequencing methods for assessing MGMT gene promotor methylation was relatively low for the cases of glioblastoma. This suggests that more reliable techniques for routine MGMT methylation study of glioblastoma remain to be developed because of quality control and assurance issues.

Role of DNA methylation of p53-Bax mitochondrial apoptosis pathway in cholangiocarcinoma / 中华临床营养杂志

Objective To study the methylation status of the promoter region of several tumor suppressor genes in p53-Bax mitochondrial apoptosis pathway and its role in cholangiocarcinoma. Methods The hypermethylation of the promoter region of tumor suppressors death-associated protein kinase (DAPK), p14, and target of methylation-associoted silencing-1 (TMS1/ASC) were detected by methylation-specific PCR. P53 gene status (exon 5-8 ) were examined by automated sequencing. The relationship between gene mutations and the biological behaviors of cholangiocarcinoma was analyzed. Results Methylation existed in at least one promoter region of tumor suppressor gene in the tumor tissues of 24 patients (66. 7% ). The frequencies of tumor suppressor gene methylation in cholangiocarcinoma were: p14 24%, DAPK 30. 6%, and TMS1/ASC 36. 1%. The frequencies of tumor suppressor gene methylation in the adjacent tissues were: TMS1/ASC 8.3% and DAPK 5.6%. DNA sequencing showed p53 gene mutation was found in 22 of 36 patients (61.1% ), and p53 gene mutation combined with the methylation of tumor suppressor was found in 14 (38.9%) patients, which was significantly correlated with pathologic biology, invasion, and differentiation ( P ＜ 0.05 ). The 1-year, 2-year, and 3-year survival rates were significantly higher in tumor-suppressing genes methylation group ( n = 4) (70%, 43 %, and 28%, respectively)than those in p53 gene mutation group (n = 14) (28%, 5%, and 0%, respectively) (χ2 =9. 060, P =0.03).Conclusions Promoter hypermethylation of p53-Bax mitochondrial apoptosis pathway is a common epigenetic event in cholangiocarcinoma. Although the methylations of TMS1/ASC and DAPK genes in the adjacent tissues are relatively low, they may be informative for the early detection of cholangiocarcinoma. P53 gene mutation combined with the methylation of tumor suppressor may be related with the pathologic biology of cholangiocarcinoma, making the latter trend to be with high malignancy and poor prognosis.

Detection of bladder cancer by accessing DNA methylation state in urine sediments / 肿瘤

Objectives: To assess DNA methylation state of the 13 genes in urine sediments and evaluate their value in diagnosis of bladder cancer. Methods: Ninety-two patients with bladder cancer, 23 patients with non tumorous urinary disease, 6 patients with neurological disease, and 7 healthy volunteers were recruited in the study. The DNA methylation state of tumor-related promoters in urine sediments was determined by Methylation specific PCR method (MSP). Results: The frequency of hypermethylation of 13 genes in 92 cases with bladder cancer was higher than that in 23 cases of bladder diseases, while all the genes were at demethylation state in the 7 healthy volunteers and 6 patients with neurological disease. The incidence of hypermethylation was significantly higher in bladder cancer compared with non tumorous urinary disease (P < 0.05). If DNA hypermethylation was used as a positive indicator, 88.0% (81/92cases) of bladder cancer could be detected. Conclusion: Assessing DNA methylation state in the promoter of tumor-related genes in urine sediments by MSP method is effective for detection of bladder cancer.

Analysis and improvement of methylation-specific polymerase chain reaction / 肿瘤研究与临床

Objective To analyze and improve Methylation-specific Polymerase Chain Reaction. Methods Methylation state of DAPK in gastric cancer was detected by this improved MSP. Results 100 % of frozen tissues and 24.1% of paraffin-embedded tissues could get either methylated production or unmethylated production. Conclusion Improved MSP is a convenient, sensitive and specific way to screen DNA methylation, especially for frozen tissues, but for paraffin-embedded tissues,it may be unsuitable.

Relationship of endometriosis with methylation status in the homeobox A10 promoter region and its protein expression / 医学研究生学报

Objective DNA methylation in the pathogenesis of endometriosis is drawing more and more attention from the medical world.This study aimed to evaluate the methylation status of the Homeobox-A10 (HOXA10) gene promoter region and the expression of the HOXA10 protein in endometriosis,and to explore the relationship of endometriosis with the aberrant methylation and expression of HOXA10 and its clinical significance.Methods The methylation-specific PCR(MSP) and the immunohistochemical SP methods were employed to detect both the methylation status of the HOXA10 promoter and its expression in the eutopic and ectopic endometrial tissues from 36 endometriosis patients,with normal endometrial tissue samples from 12 healthy women as controls.Results Hypermethylation of the HOXA10 promoter region was present in 38.9% (14/36) of the ectopic endometrial tissue samples and 25.0% (9/36) of the eutopic endometrial tissue samples,all in endometriosis of stages Ⅲ and Ⅳ,but no hypermethylation was found in the endometrial tissues of the normal controls,with significant differences between the two groups (P

Effects of homocycteine on methylation status and mRNA expression of TNFSF4 gene in macrophages in vitro / 第三军医大学学报

Objective To investigate the effects of homocysteine (Hcy) on methylation status and mRNA expression of TNF superfamily member 4 (TNFSF4) gene in THP-1 macrophages. Methods Cultured THP-1 monocytes were induced to macrophages by 0.1 ?mol/L PMA treatment for 72 h, then the differentiated THP-1 macrophages were incubated with homocysteine (50-1 000 ?mol/L) for 24, 48 or 72 h. The status of methylation of TNFSF4 gene in THP-1 macrophages was analyzed by methylation specific polymerase chain reaction (MS-PCR).The expression level of TNFSF4 mRNA was determined by RT-PCR. Results The MS-PCR results showed that the unmethylated bands gradually became stronger, and the methylated bands gradually became weaker along with the prolongation of treatment time and the increased Hcy concentrations. The 72-hour treatment with Hcy induced a complete demethylation in the promotor region of TNFSF4 gene, where left the only unmethylated bands. Meanwhile, the TNFSF4 mRNA expression was also increased in a dose-dependent manner. Conclusion Hcy may contribute to atherogenesis by inducing demethylation in the promotion region of the TNFSF4 gene and increasing TNFSF4 expression in THP-1 macrophages.