前列腺癌患者外周血游离DNA GSTP1基因甲基化检测及其意义

发布时间：2018-08-07 09:51

【摘要】：目的:DNA甲基化是一种调节基因表达的重要机制,在肿瘤发生、发展中起重要作用。研究表明,在肿瘤诊断、预后及疗效判断方面,DNA甲基化是一种有潜在应用价值的肿瘤标志物。本研究运用自行建立的实时荧光定量PCR甲基化检测方法,检测前列腺癌患者外周血游离DNA中谷胱苷肽-S-转移酶P1(GSTP1)基因启动子区域甲基化表达情况,探讨其在前列腺癌早期诊断中的价值。方法:收集入院治疗的患者的外周血样本,每位患者各2管,约2ml/管,置于-80℃超低温冰箱冻存。其中符合入组标准的样本分别为:行前列腺癌内分泌治疗患者58例作为A组,患者年龄波动于61~82岁,平均年龄72岁;因“排尿困难,发现PSA升高”入院就诊的患者42例作为B组,其中23例诊断为前列腺癌(B1),19例诊断为非前列腺癌(B2),B组年龄波动于55~78岁,平均年龄70岁;非前列腺疾病患者34例作为C组,PSA在正常参考值范围内,为同期因尿道狭窄、肾结石、肾囊肿等良性疾病就诊的男性患者,其年龄波动于58~76岁,平均年龄68岁。运用甲基化特异性PCR法对三组患者GSTP1基因的表达情况进行定性检测,运用定量PCR检测GSTP1基因的甲基化相对表达量,统计分析其与前列腺癌患者主要临床病理特征之间的关系。结果:1.A组患者外周血游离DNA中GSTP1基因的甲基化相对表达量(M/U)为0.330973310±0.1848315242,B2组患者的相对表达量为1.585767684±0.8382501872,C组患者的相对表达量为2.354086941±2.1664719163,Welch检验P=0.0000.05,所以A、B2、C三组总体均数不全相等。两两比较,A、B2两组总体均数间存在显著差异(P=0.000);A、C两组总体均数间存在显著差异(P=0.000);B2、C两组总体均数间不存在显著差异(P=0.230)。可认为A组患者GSTP1基因甲基化比值低于B2、C两组。2.B1组患者GSTP1基因的甲基化相对表达量(M/U)为0.304547000±0.1905957866,与B2组比较分析,Welch检验P=0.0000.05,两组总体均数间存在显著差异。3.A组患者与B1组患者GSTP1基因的甲基化相对表达量比较分析,采用独立样本t检验,P=0.8060.05,两组总体均数间无显著差异。4.前列腺癌患者GSTP1基因甲基化程度与相应PSA相关性分析,Kendall’tau_b、Spearman’rho两种非参数方法检验,Kendall相关系数为0.198,Spearman相关系数为0.263,均为P0.05,提示前列腺癌患者外周血游离DNA中GSTP1基因甲基化相对表达量与PSA相关性不显著(相关系数小于0.5)。5.Gleason评分为5~7分和8~10分的GSTP1基因甲基化程度总体均数间无显著差异(P=0.930),危险度分级为中低危和中高危的GSTP1基因甲基化程度总体均数间无显著差异(P=0.241)。结论:1.前列腺癌患者和前列腺良性病变患者外周血游离DNA中GSTP1基因均存在甲基化和非甲基化表达;2.前列腺癌患者外周血游离DNA中GSTP1基因甲基化表达量比值(M/U)较低,而良性病变患者(BPH等)的甲基化比值较高,推测是机体对肿瘤产生的反馈性保护作用;3.内分泌治疗对前列腺癌患者外周血游离DNA中GSTP1基因甲基化相对表达量无影响;4.前列腺癌患者外周血游离DNA中GSTP1基因甲基化相对表达量与PSA相关性不显著(r0.5),可将其作为筛查前列腺癌的独立生物标记物;5.检测患者外周血GSTP1基因甲基化相对表达量,虽然对前列腺癌的Gleason评分和危险度分级无预测作用,但对提高前列腺癌早期诊断准确率有一定价值。
[Abstract]:Objective: DNA methylation is an important mechanism for regulating gene expression and plays an important role in the occurrence and development of tumor. The study shows that DNA methylation is a potentially valuable tumor marker in the diagnosis, prognosis and evaluation of the tumor. This study uses a real-time fluorescence quantitative PCR methylation detection method established by ourselves to detect the tumor. The methylation of the promoter region of the glucocysteinase -S- transferase P1 (GSTP1) gene in the peripheral blood free DNA of the prostate cancer patients was measured and the value of it was discussed in the early diagnosis of prostate cancer. Methods: the peripheral blood samples of the patients treated by the hospital were collected, each of the patients was 2 tubes, about 2ml/ tube, and stored at -80 C cryopreservation. The standard samples of the group were as follows: 58 patients with prostate cancer endocrine therapy were treated as A group. The age of the patients was 61~82 years old and the average age was 72 years old; 42 cases were treated as B group because of "dysuria and PSA rise", of which 23 were diagnosed as prostate cancer (B1), 19 were diagnosed as non prostate cancer (B2), and the B group fluctuated in 55~7. 8 years old, the average age of 70 years, 34 cases of non prostatic disease patients as C, PSA in the normal reference range, for the same period of urethral stricture, kidney stones, renal cysts and other benign diseases in male patients, whose age fluctuates at the age of 58~76 and the average age of 68 years. The expression of GSTP1 gene in three groups of patients by methylation specific PCR The relative expression of methylation of GSTP1 gene was detected by quantitative PCR, and the relationship between the main clinicopathological features of the prostate cancer patients was statistically analyzed. Results: the relative expression of GSTP1 gene (M/U) was 0.330973310 + 0.1848315242 in the peripheral blood free DNA of the 1.A group, and the relative expression of the B2 group was 1.5857. 67684 + 0.8382501872, the relative expression of patients in group C was 2.354086941 + 2.1664719163, Welch test P=0.0000.05, so A, B2, C three groups were not all equal. 22 compared, A, B2 two groups there were significant differences (P=0.000), A, C two groups there were significant differences (P=0.000); two groups of the total number of two groups did not exist obvious There was a difference (P=0.230). The methylation ratio of GSTP1 gene in group A patients was lower than that of B2, and the relative expression of GSTP1 gene methylation (M/U) in group C two was 0.304547000 + 0.1905957866, compared with the B2 group and Welch test P=0.0000.05. There were significant differences in the methylation of the two groups. Relative expression analysis, using independent sample t test, P=0.8060.05, there was no significant difference between the total number of the two groups. The degree of methylation of GSTP1 gene in.4. prostate cancer patients was related to the corresponding PSA correlation analysis, Kendall 'tau_b, Spearman' Rho two non parametric methods, the Kendall correlation coefficient was 0.198, the Spearman correlation coefficient was 0.263, all P. 0.05, the correlation between the relative expression of GSTP1 gene methylation and PSA in the peripheral blood free DNA of the prostate cancer patients was not significant (the correlation coefficient was less than 0.5). There was no significant difference between the GSTP1 gene methylation degree of the 5~7 and 8~10 scores (P=0.930), and the risk grade was the middle and low risk and the middle risk of the GSTP1 gene methylation. There was no significant difference between the total number of degrees (P=0.241). Conclusion: 1. the GSTP1 genes in peripheral blood free DNA of the prostate cancer patients and benign prostatic lesions all have methylation and non methylation expression; 2. the GSTP1 gene methylation expression ratio (M/U) in the peripheral blood free DNA of the prostate cancer patients is lower than that of the benign disease patients (BPH and so on). The higher the ratio of the substrate, it is presumed to be the feedback protective effect of the body on the tumor; 3. the endocrine therapy has no effect on the relative expression of GSTP1 gene methylation in the peripheral blood free DNA of the patients with prostate cancer; 4. the relative expression of the methylation in the free DNA of the peripheral blood of the prostate cancer patients is not significantly associated with the PSA (r0.5). In order to screen the independent biomarkers of prostate cancer, 5. the relative expression of GSTP1 gene methylation in peripheral blood of the patients has no predictive effect on the Gleason score and risk classification of prostate cancer, but it is of certain value in improving the accuracy of early diagnosis of prostate cancer.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R737.25

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