乳腺癌miR-34a表达及其对化疗耐药分子机制影响探讨

Expression of miR-34a in breast cancer and its molecular mechanism of drug resistance

目的 miR-34a在多种肿瘤有异常表达并参与肿瘤的多药耐药。本研究探讨乳腺癌miR-34a表达及其参与乳腺癌化疗耐药的分子机制。方法选择南昌市第三医院2008-08-01—2010-11-30收治的原发性乳腺癌患者113例,采用qRT-PCR方法检测乳腺癌细胞及乳腺癌组织miR-34a表达,对比分析耐药细胞与亲代细胞的表达差异,分析miR-34a表达与乳腺癌临床病理特征及复发转移的关系。miR-34amimic转染乳腺癌耐药细胞株MDR-MCF-7,MTT检测细胞耐药指数的改变,流式细胞方法分析细胞周期分布的变化,qRT-PCR和蛋白质印迹法检测p53、Bcl-2、TOP-2a、CCND1和Notch1的表达变化。结果耐药细胞株MDR-MCF-7较其亲代乳腺癌细胞MCF-7的miR-34a表达降低了73.8%。耐药细胞株MDR-MCF-7转染miR-34amimic后,细胞对多柔比星、环磷酰胺、多西他赛和5-FU的IC_(50)均有不同程度的降低,出现明显的G_0/G_1期阻滞,处于分裂期(G2/M)的细胞数明显减少。miR-34a可明显下调靶基因Bcl-2、CCND1和Notch1的mRNA和蛋白表达水平,而p53和TOP-2a变化均不明显。乳腺癌组织miR-34a的表达与淋巴结转移状态、Bcl-2、CCND1、HER2和Notch1表达相关,而与月经状态、组织学分级、临床分期、ER、PR、p53和TOP-2a的表达无关。复发乳腺癌组织的miR-34a表达水平明显低于无复发转移的乳腺癌组织,P<0.01。结论 miR-34a参与乳腺癌的化疗耐药,其可能通过下调靶基因Bcl-2、CCND1和Notch1的表达来抑制细胞耐药,乳腺癌组织miR-34a低表达是预后不良的标志。

OBJECTIVE The objective of this study was to explore the expression of miR-34a in patients with breast cancer and its molecular mechanism of drug resistance in breast cancer. METHODS Totally 113 patients with breast cancer who were diagnosised from Aug. 1,2008 to Nov. 30,2010 in Third Hospital of Nanchang was choosen. The expression of the miR-34a of the cells and tissues of breast cancer was detected by qRT-PCR. Compared the different expression of the resistant cell and parental cell and analyzed the relationship of the clinicopathologic characteristics and recurrence,metastasis of patients. To analyze the transfection of the miR-34a mimic for MDR-MCF-7, the change of drug resistance index was detected by MTT, cell cycle percentage was detected by flow cytometry, the expression of p53, Bcl-2, TOP-2a,CCND1 and Notch1 was detected by qRT-PCR and Western Blotting. RESULTS The expression of miR-34a of MDR-MCF-7 was 73.8% lower than the MCF-7 of parental cell. After the transfection of the miR-34a mimic for MDR- MCF-7,the IC50 of adriamycin,cyclophosphamide,docetaxel and 5-fluorouracil was lower. The G0/G1 period was retardant, the G2/M period cell number also decreased, miR-34a could lower the expression of the expression of mRNA and protein for Bcl-2,CCND1 and Notchl, the changes of p53 and TOP-2a were not obvious. The expression of miR-34a was related to the expression of mRNA and protein for Bcl-2,CCND1, HER2 and Notchl, but it had nothing to do with meno- pausal status,histologic type, Staging, ER, PR, p53 and TOP-2a. The expression of miR 34a for patients with recurrent was low than patients without recurrence and metastasis（P〈0.01）. CONCLUSIONS miR 34a participate in the molecu lar mechanism of drug resistance in breast cancer,and it can lower the expression of the target gene to inhibit cell drug resistance. The sign of the poor prognosis for breast cancer is the lower expression of miR 34a.