| Objective:Differential diagnosis of pheochromocytoma (PHEO) and paraganglioma (PGL) between benign and malignant and the effective treatment of malignant PHEO and PGL are the unsolved problems in the research field of neuroendocrine tumors today. In addition to genetic alterations, epigenetic change is another important research field in tumorigenesis and pathogenesis of disease. The epigenetic research of PHEO and PGL has just begun in other countries. The study was undertaken to detect the promoter methylation status of ten genes using epigenetic method, including p16INK4a which is related to regulation of cell cycles, hMLH1, MGMT, and BRCA1 which are related to DNA repair, RASSF1A which is a important molecule in the ras signal pathway, DcR2 which is related to cell apoptosis, SDHB, SDHD, and VHL whose mutation are involved in pathogenesis of PHEO and PGL, LRP1B which was down-regulated in PHEO in our previous study, and to look for CpG island methylation phenotype(CIMP) in PHEO and PGL, and to explore the relationship between gene promoter methylation and malignant behavior or other clinical characteristics of patients.Methods:1. Tissue DNA was extracted from 24 cases of PHEO (10 cases were malignant and 14 cases were benign),29 cases of PGL (14 cases were malignant and 15 cases were benign), and 7 cases of normal adrenal medulla with EZNA TM DNA/RNA Isolation Kit.2. Methylation status of p16INK4a, RASSF1A, DcR2, MGMT, SDHB, SDHD, VHL, LRP1B, BRCA1, hMLH1 genes was detected using methylation specific PCR method.3. Analyze the relationship between the gene promoter methylation status and the clinical data (such as malignant behavior) from PHEO and PGL patients, and investigate the role of the gene promoter methylation status in the pathogenesis of PHEO and PGL.Results:1. Methylation of p16INK4a,RASSF1A, MGMT and DcR2 genes in PHEO and PGL1) Methylation of p16INK4a, RASSF1A, MGMT and DcR2 genes were detected in 53 cases of PHEO and PGL. Detection rate of methylation was 52.8%,52.8%,35.8% and 60.4%, respectively. In PHEO, detection rate of methylation of these four genes were 58.3%(14/24),58.3%(14/24),41.7%(10/24) and 54.2%(13/24) respectively. The number of malignant cases were 8 cases (33.3%),6 cases (25.0%),5 cases (20.8%), and 5 cases (20.8%), respectively. In PGL, detection rate of methylation of these four genes were 48.3%(14/29),48.3%(14/29), 31.0%(9/29) and 65.6%(19/29), respectively. The number of malignant cases werell cases(37.9%),10 cases(34.5%),6 cases(20.7%), and 14 cases(48.3%), respectively.2) Detection rate of p16INK4a methylation in benign PHEO, malignant PHEO, benign PGL and malignant PGL was 42.9%(6/14),80.0%(8/10),20.0%(3/15)and 8.6% (11/14), respectively.3) Detection rate of RASSF1A methylation in benign PHEO, malignant PHEO, benign PGL and malignant PGL was 57.1%(8/14),60.0%(6/10),26.7%(4/15), and 71.4%(10/14), respectively.4) Detection rate of MGMT methylation in benign PHEO, malignant PHEO, benign PGL and malignant PGL was 35.7%(5/14),50.0%(5/10),20.0%(3/15), and 42.9%(6/14),respectively.5) Detection rate of DcR2 methylation in benign PHEO, malignant PHEO, benign PGL and malignant PGL was 57.14%(8/14),50.00%(5/10),33.33%(5/15) and 100.0%(14/14),respectively.6) At least one methylated gene was detected in malignant PHEO and PGL. There was no methylated genes were found in 8 benign cases (1case of PHEO,7 cases of PGL). CpG island methylator phenotype (CIMP) positive tissue was found in 19 cases, including 5 cases of malignant PHEO,4 cases of benign PHEO,9 cases of malignant PGL, and 1 case of benign PGL.2. The clinical significance of methylation of p16INK4a, RASSF1A, MGMT and DcR2 genes in PHEO and PGL1) The level of 24h urinary norepinephrine (NE) and blood NSE from malignant PGL patients was significantly higher than benign ones (p=0.017,0.034).2) The level of 24h urinary epinephrine (E) from PHEO patients with p16INK4a methylation was markedly higher than those without p16INK4a methylation (p=0.014). 3) p16INK4a methylation was more common in malignant PGL than in benign PGL, the difference was statistically significant (p=0.002). p16INK4a methylation was found in younger patients with PGL, but the difference was not significant (p=0.085). RASSF1A methylation were more common in malignant PGL than in benign PGL, the difference was statistically significant (p=0.016). DcR2 methylation more common in younger patients, there was a significant difference (p=0.003).4) Methylation index (MI) was significantly higher in malignant patients than in benign patients with PGL (p<0.001). CIMP was more common in malignant PGL than in benign PGL (p=0.002)3. The relationship of methylation of p16INK4a, RASSF1A, MGMT and DcR2 genes in PHEO and PGL1) In PHEO, RASSF1A methylation was correlated with DcR2 methylation (r=0.410, p<0.05).2) In PGL, methylation between DcR2 and p16INK4a gene was correlated (r=0.556, p<0.01), RASSF1A methylation was correlated with DcR2 and MGMT methylation (r=0.396, p<0.05 and r=0.411, p<0.05, respectively).4. Methylation of SDHB, SDHD, VHL, LRP1B, BRCA1 and hMLH1genes in PHEO and PGLWe could not detect methylation of any other six genes in all of the tissues of PHEO and PGL.Conclusions:1. Methylation of at least one of the four genes (p16INK4a RASSF1A, DcR2, and MGMT) was detected in 84.9% of PHEO and PGL, which indicated that gene methylation was involved in tumorigenesis and tumor development.2. p16INK4a, RASSF1A and DcR2 methylation were associated with malignant behavior of PGL. Detection of promoter methylation status of these genes may help to identify the benign and malignant PGL.3. Methylation index and CpG island methylator phenotype (CIMP) were associated with malignant behavior of PGL, suggesting that malignant PGL tumors have a higher level of multiple gene promoter methylation. Detection of multiple gene promoter methylation status has implications for distinguishing the benign and malignant PGL. ObjectiveIn this study, we collected the plasma of patients with PHEO and PGL whose tumor tissue were conducted to detect DNA methylation in the first part of study, in an attempt to detect p16INK4a and RASSF1A gene methylation status using seminested methylation-specific MSP, and to analysis the relationship between methylation status in plasma and tissue DNA so as to provide experimental basis for early differential diagnosis of benign and malignant cases.Methods1. DNA was extracted from the plasma from patients with malignant PGL (n=8), benign PGL (n=6), malignant PHEO (n=7), and benign PHEO (n=8) using Qiagen DNA blood mini kit.2. p16NK4a and RASSF1A gene methylation status in plasma DNA was detected by semi-nested methylation-specific PCR. Gene promoter methylation in plasma was compared with that in tissue, and the relationship between gene methylation status and the clinical characteristics was analyzed.Results1. The detection rates of p16INK4a gene methylation in plasma and tissue DNA which derived from 29 cases of PHEO/PGL was 44.83%(13/29) and 55.17%(16/29), respectively. There was no significant difference in the detection rates of p16INK4a gene methylation between plasma and tissue DNA. Correlation analysis showed that there was a positive correlation in p16INK4a gene promoter methylation between plasma and tumor tissue (r=0.813, p<0.001).2. The detection rates of RASSF1A gene methylation in plasma and tissue DNA which derived from 29 cases of PHEO/PGL was 31.03%(9/29) and 44.83%(13/29), respectively. There was no significant difference in the detection rates of RASSF1A gene methylation between plasma and tissue DNA. Correlation analysis showed that there was a positive correlation in RASSF1A gene promoter methylation between plasma and tumor tissue (r=0.694, p<0.001).3. There was no significant difference between p16INK4a gene methylation and different clinical characteristics including sex, age, hereditary, location, tumor number, tumor size, and Ki-67% (p> 0.05). Like tumor tissue, p16INK4a gene methylation was more common in malignant patients, the difference was statistically significant (p<0.05).4. There was no significant difference between RASSF1A gene methylation and different clinical characteristics including sex, age, tumor nature, tumor number, tumor size, secretion levels of 24h urinary catecholamine, and Ki-67%(p> 0.05).Conclusionsp16INK4a and RASSF1A gene promoter methylation in plasma free DNA was detected by semi-nested MSP, it had a positive correlation with the methylation status of the genes in tumor tissues, suggesting that the detection of gene promoter methylation status may provide a clinical application for early differential diagnosis of benign and malignant cases. ObjectiveIn the first and second part of the study, p16INK4a and RASSF1A methylation in plasma and tissue DNA was detected, so the purpose of this study was to investigate p16INK4a and RASSF1A mRNA expression in PHEO and PGL and normal adrenal medulla, and to explore the relationship between mRNA expression and clinical characteristics of patients or the gene promoter methylation in order to understand the role of p16INK4a and RASSF1A gene in pathogenesis of PHEO and PGL.Methods1. Tissue total RNA was extracted from malignant PHEO (n=10), benign PHEO (n= 14), malignant PGL (n=14), benign PGL (n=15), and normal adrenal medulla (n=7) with EZNA TM DNA/RNA Isolation Kit.2. Real-time quantitative PCR was carried out to analyze gene expression of p16INK4a and RASSF1A in different tissues. The expression levels of the target genes were relatively quantified usingβ-actin as internal control.3. The relationship between mRNA expression levels of p16INK4a, RASSF1A and clinical characteristics of patients with PHEO and PGL, or gene promoter methylation were analyzed.Results:1. The mRNA expression of p16INK4a in PHEO and PGL1) p16INK4a mRNA expression levels in malignant PHEO, benign PHEO, malignant PGL, and benign PGL were significantly lower than that in normal adrenal medulla (p<0.01);2) The difference of p16INK4a mRNA expression among malignant PHEO, benign PHEO, malignant and benign PGL were not statistically significant (vs malignant PGL, p=0.088,0.213,0.367).2. The mRNA expression of RASSF1A in PHEO and PGL1) RASSF1A mRNA expression in malignant PHEO, benign PHEO, malignant PGL, and benign PGL were significantly lower than that in normal adrenal medulla (p<0.05); 2) The difference of RASSF1A mRNA expression among malignant PHEO, benign PHEO, malignant and benign PGL was not statistically significant (vs malignant PGL,p=0.314,0.732,0.876).3. The relationship between p16INK4a and RASSF1A mRNA expression and clinical features of patients with PHEO and PGL.1) There was no significant difference between p16INK4a mRNA expression and clinical characteristics such as gender, age, tumor location, number, size, and Ki-67%(p>0.05).2) There was also no significant difference between RASSF1A mRNA expression and different clinical characteristics (p> 0.05).4. The relationship between and p16INK4a and RASSF1A mRNA expression and gene promoter methylation1) The difference of p16INK4a mRNA expression between PHEO with methylated p16INK4a and PHEO with unmethylated gene, or between PGL with methylated p16INK4a and PGL with unmethylated gene, was no significant (p> 0.05). There was no significant correlation between p16INK4a mRNA expression and promoter methylation status (p> 0.05).2) The difference of RASSFIA mRNA expression between PHEO with methylated RASSF1A and PHEO with unmethylated gene or between PGL with methylated RASSF1A and PGL with unmethylated gene was no significant (p> 0.05). There was no significant correlation between RASSFIA mRNA expression and promoter methylation status (p> 0.05).Conclusions:p16INK4a and RASSFIA mRNA expression were down-regulated in PHEO and PGL, but there was no significant correlation between p16INK4a and RASSFIA mRNA expression and promoter methylation status, indicating that gene promoter methylation is one of the inactivation mechanisms for p16INK4a and RASSF1A genes, and there were other mechanism except DNA methylation to inactivate gene expression, such as loss of heterozygosity, other epigenetic abnormalities (his tone acetylating, miRNA interference) and so on.
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