COBRA和QM-PCR法检测乳腺癌中PANDAR基因DNA甲基化

发布时间：2018-12-15 02:13

【摘要】：[目的]通过联合亚硫酸氢钠的限制性内切酶分析法技术(COBRA),定性检测乳腺癌及癌旁正常组织中PANDAR基因DNA甲基化的情况,再通过荧光定量甲基化PCR (QM-PCR)技术对乳腺癌和癌旁正常组织中PANDAR基因DNA甲基化数值进行定量检测并分析,探讨PANDAR基因在乳腺癌中DNA甲基化的临床意义。[方法]收集乳腺癌及癌旁正常组织30对,所采集癌旁正常乳腺组织距离肿块至少不小于5cm,并且可在不同象限取材,分别提取DNA,采用联合亚硫酸氢钠的限制性内切酶分析法(COBRA)检测乳腺癌组织和癌旁组织的PANDAR基因DNA甲基化的大致情况,采用QM-PCR技术对乳腺癌组织和癌旁正常组织PANDAR基因DNA甲基化的程度定量检测,使用spssl7.0软件对实验数据进行处理,结果行统计学分析。[结果]1. COBRA技术检测30例乳腺癌组织中以及癌旁正常组织中,经Bsp119I酶酶切后乳腺癌组织中PANDAR出现DNA甲基化程度高于50%的有28例,甲基化程度低于50%的有2例。P=1；经HinfI酶酶切后乳腺癌组织中PANDAR出现DNA甲基化程度高于50%的有30例,甲基化程度低于50%的有0例。P=10.05,差异均无统计学意义。2.经过荧光定量甲基化PCR (QM-PCR)技术检测30例乳腺癌组织中以及癌旁正常组织中,PANDAR基因出现DNA甲基化的比例为74%,癌旁正常组织出现DNA甲基化的比例为68%, P=0.00690.05,差异有统计学意义。[结论]1.在30例乳腺癌组织中可见PANDAR基因启动子CpG岛甲基化水平高于癌旁正常组织。以上结果提示：DNA甲基化与PANDAR基因在乳腺癌组织中高表达是有相关性的。2.乳腺癌组织中PANDAR基因DNA甲基化的水平较癌旁正常乳腺组织明显升高,PANDAR基因启动子CpG岛的甲基化状态与该基因在乳腺癌组织中高表达有关,提示PANDAR基因DNA甲基化可能是一个较有价值的分子标志,其甲基化水平可能会成为检测乳腺癌的新的特异性指标,在乳腺癌的发生过程中起重要作用,并可能成为治疗乳腺癌的特异性潜在生物靶点。
[Abstract]:[objective] to qualitatively detect the DNA methylation of PANDAR gene in breast cancer and adjacent normal tissues by (COBRA), combined with sodium bisulfite restriction endonuclease assay. The value of DNA methylation of PANDAR gene in breast cancer and adjacent normal tissues was quantitatively detected and analyzed by fluorescence quantitative methylation PCR (QM-PCR). The clinical significance of PANDAR gene in DNA methylation in breast cancer was discussed. [methods] A total of 30 pairs of breast cancer and adjacent normal tissues were collected. The distance between the adjacent normal breast tissues and the mass was not less than 5 cm, and DNA, could be extracted from different quadrants. The DNA methylation of PANDAR gene in breast cancer tissues and adjacent tissues was detected by restriction endonuclease assay (COBRA) combined with sodium bisulfite. The degree of DNA methylation of PANDAR gene in breast cancer tissues and adjacent normal tissues was quantitatively detected by QM-PCR technique. The experimental data were processed by spssl7.0 software. The results were statistically analyzed. [result] 1. COBRA technique was used to detect DNA methylation in 30 breast cancer tissues and adjacent normal tissues. 28 cases of breast cancer tissues with DNA methylation more than 50% and 2 cases with less than 50% DNA methylation were detected by Bsp119I enzyme digestion. There were 30 cases with PANDAR methylation higher than 50% and 0 cases with less than 50% DNA methylation in breast cancer tissues digested by HinfI enzyme. There was no significant difference between the two groups. 2. By fluorescence quantitative methylation PCR (QM-PCR) technique, the percentage of DNA methylation in 30 breast cancer tissues and adjacent normal tissues was 74%, and that of DNA methylation in adjacent normal tissues was 68%. The difference was statistically significant. [conclusion] 1. In 30 cases of breast cancer, CpG island methylation level of PANDAR promoter was higher than that of adjacent normal tissues. These results suggest that DNA methylation is associated with the overexpression of PANDAR gene in breast cancer. The level of DNA methylation of PANDAR gene in breast cancer tissues was significantly higher than that of adjacent normal breast tissues. The methylation status of CpG island of PANDAR gene promoter was related to the high expression of PANDAR gene in breast cancer tissues. The results suggest that DNA methylation of PANDAR gene may be a valuable molecular marker, and its methylation level may become a new specific marker for detection of breast cancer and play an important role in the pathogenesis of breast cancer. It may be a potential biological target for the treatment of breast cancer.
【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R737.9

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