微RNA-34a在乳腺癌中的表达及其基因启动子甲基化

Expression and promoter methylation of microRNA-34a gene in human breast cancer

目的 :探讨人乳腺癌组织中微RNA-34a(micro RNA-34a,mi R-34a)的表达及其临床意义,并分析mi R-34a基因启动子甲基化状态与成熟mi R-34a表达及患者生存的相关性。方法 :采用Taq Man探针-实时荧光定量PCR法检测93例乳腺癌组织及5例良性乳腺疾病标本(作为对照)中成熟mi R-34a的表达。运用Epi Tect Fast FFPE Bisulfi te试剂盒对石蜡包埋乳腺癌组织的基因组DNA进行抽提及亚硫酸盐修饰,然后采用甲基化特异性PCR法检测mi R-34a基因启动子的甲基化状态。统计学分析mi R-34a基因启动子甲基化状态与mi R-34a表达量之间的关系,并用生存曲线分析其预后意义。结果 :乳腺癌组织中成熟mi R-34a的表达水平比良性对照组明显降低(P=0.006)。mi R-34a表达水平与肿瘤大小(r=-0.312,P=0.021)、淋巴结转移(r=-0.378,P=0.004)和肿瘤分级(r=-0.341,P=0.011)均呈负相关性,而与雌激素受体(estrogen receptor,ER)、孕激素受体(progesterone receptor,PR)和人表皮生长因子受体2(human epidermal growth factor receptor-2,HER-2)等水平均不相关(P均>0.05)。mi R-34a基因启动子非甲基化组的成熟mi R-34a表达水平明显高于甲基化阳性单一模式组和甲基化阳性混合模式组(P值均<0.001)。与非甲基化组和甲基化阳性混合模式组相比,mi R-34a基因甲基化阳性组的乳腺癌患者无复发生存期(P<0.001,P=0.019)及总生存期(P=0.002,P<0.001)均明显缩短。结论 :在部分乳腺癌患者中,mi R-34a基因甲基化可导致mi R-34a表达下调。mi R-34a基因的甲基化状态与乳腺癌的复发及不良预后相关。

Objective： To investigate the expression and clinical significance of microRNA-34a （miR-34a） in human breast cancer tissues, and to analyze the correlation of methylation status of miR-34a gene promoter with the expression of mature miR-34a and the survival of patients with breast cancer. Methods： The expression level of mature miR-34a in breast cancer tissues （n = 93） and benign breast lesions （n = 5, as the control） was detected by raqMan probe-based real-time fluorescent quantitative PCR （TaqMan MicroRNA Assay Kit）. The bisulfite conversion and purification of genomic DNA from each formalin-fixed and paraffin-embedded breast cancer samples were performed by EpiTect Fast FFPE Bisulfite Kit. The methylation status of miR- 34a gene promoter in breast cancer samples was assessed by methylation-specific PCR. The relationship between miR-34a expression and miR-34a gene methylation was statistically analyzed. The prognostic value of rniR-34a gene methylation was analyzed by Kaplan-Meier curve. Results： The expression level of miR-34a in breast cancer was significantly lower than that in benign breast lesions （P = 0.006）. The expression of miR-34a was negative correlated with tumor size （r = -0.312, P = 0.021）, lymph node metastasis （r = -0.378, P = 0.004） and tumor grade （r = --0.341, P = 0.011）. However, the expression of miR-34a was not associated with estrogen receptor （ER）, progesterone receptor （PR） or human epidermal growth factor receptor-2 （HER-2） （all P 〉 0.05）. The level of mature miR-34a in unmethylated group was significantly higher than that in methylated/unmethylated group or methylated group （both P 〈 0.001）. Compared with unmethylated group and methylated/unmethylated group, the recurrence-free survival （P 〈 0.001, P = 0.019） and overall survival （P = 0.002, P 〈 0.001） of patients with breast cancer were significantly reduced in rniR-34a gene methylated group. Conclusion： In some patients with breast cancer, the methylation of miR-34a gene may lead to the down-regulation of mature miR-34a expression. Furthermore, the methylation status of miR-34a gene is associated with the risk of tumor recurrence and poor prognosis in patients with breast cancer.