乳腺癌组织与细胞株雄激素受体生物学作用分子机制探讨

Expression and molecular mechanisms of androgen receptors in breast cancer tissue and cells

目的乳腺癌是激素依赖性肿瘤,除雌激素外,雄激素通过与雄激素受体(androgen receptor,AR)相结合在乳腺癌的发生发展中也发挥重要作用,但其机制以及对乳腺癌的意义并不明确,本研究通过探讨乳腺癌组织及细胞中AR的表达及分子机制,为乳腺癌的治疗寻找潜在的新靶点。方法选取2013-01-01-2016-10-01郑州大学附属郑州中心医院经手术治疗的乳腺癌原发肿瘤患者组织标本150例,免疫组化法检测150例乳腺癌组织中AR蛋白的表达;蛋白质印迹法及RT-PCR法检测不同病理分级中AR蛋白表达;蛋白质印迹法、RT-PCR及免疫荧光的方法检测AR蛋白在转移及非转移性乳腺癌细胞株中的表达。应用miRanda软件平台预测可能作用于AR mRNA 3′UTR保守位点的miRNA。使用Real Time RT-PCR分析在非转移和转移乳腺癌细胞株中可能靶向AR mRNA的miRNA,并分析不同病理分级乳腺癌组织中可能靶向AR mRNA的miRNA。结果 AR蛋白在乳腺癌组织中表达与组织学分级呈负相关,r=-0.473,P<0.001;Ⅰ、Ⅱ级与Ⅲ级相比,差异有统计学意义,χ2=38.233,P<0.001。蛋白质印迹法检测结果显示,乳腺癌组织Ⅰ、Ⅱ和Ⅲ级积分光密度值(integrated option density,IOD)分别为2.291±0.265、0.229±0.030和0.277±0.043,Ⅰ级与Ⅱ、Ⅲ级比较差异有统计学意义,z=-3.794,P<0.001,即Ⅱ、Ⅲ级表达明显降低。Ⅱ级与Ⅲ级比较差异无统计学意义,z=-1.973,P=0.052。RT-PCR结果显示,AR蛋白在乳腺癌组织Ⅰ、Ⅱ和Ⅲ级中相对表达量分别为1.00±0.11、0.53±0.15和0.59±0.12,Ⅱ(t=6.177,P=0.003)、Ⅲ级(t=4.162,P=0.014)显著低于Ⅰ级,Ⅱ与Ⅲ级比较差异无统计学意义,t=-1.628,P=0.179。即病理分级Ⅰ级中AR蛋白及mRNA水平显著高于Ⅱ和Ⅲ级。非转移乳腺癌细胞株(BT-549)和转移乳腺癌细胞株(MDA-MB-23)中,AR蛋白的IOD比值分别为1.980±0.101和0.926±0.072,t=14.718,P<0.001;BT-549和MDA-MB-231细胞系AR的mRNA相对表达量分别为1.000±0.110和0.020±0.012,t=107.245,P<0.001。即MDA-MB-231细胞株中AR蛋白及mRNA水平显著低于BT-549细胞株。RT-PCR分析结果显示,转移乳腺癌细胞株中miR-873和miR-185表达高于非转移乳腺癌细胞株,并且病理分级Ⅰ级中miR-873和miR-185表达低于Ⅱ和Ⅲ级,但仅miR-873差异有统计学意义,F=11.065,P=0.010。结论乳腺癌组织及细胞中AR表达与组织学分级及有无转移呈负相关,其机制可能与高级别乳腺癌组织中miR-873上调进而负调控AR mRNA有关。

OBJECTIVE Breast cancer is known as a hormone-responsive cancer. Apart from estrogen, androgen also plays a critical role in the occurrence and development of breast cancer by combining with androgen receptor（AR）, but its mechanism and the significance for the prognosis of breast cancer are not clear. The aim of this study is to investigate the expression and molecular mechanism of AR in breast cancer tissues and cells, and to find new targets for the treatment of breast cancer. METHODS Immunohistochemical technique was used to detect AR expression in 150 cases of breast cancers. Western Blot and RT-PCR were used to assess the expression of AR in different pathological grades of breast cancers. The expression of AR in metastatic and non-metastatic breast cancer cells was detected by immunofluores- cence, Western Blot and RT PCR. MiRanda bioinformatics＇ prediction software was used to predict the possible miRNAs whose target gene is AR. Real Time RT-PCR was used to analyze miRNAs that could target AR mRNA in non-metastatic and metastatic breast cancer cells and in different pathological grading in breast cancer tissues. RESULTS The expression of AR protein was negatively correlated with histological grade in breast cancers （r=-0. 473 ,P〈0. 001） and there was a significant statistical difference between level Ⅰ, Ⅱ and Ⅲ （X2 = 38. 233, P〈0. 001）. Western Blot results showed IOD ratio of grade Ⅰ, grade Ⅱ and III of breast cancer was 2. 291±0. 265, 0. 229±0. 030 and 0. 277±0. 043, respectively, there were significant statistical differences between level Ⅰ , Ⅱ and Ⅱ （z=-3. 794,P〈0. 001）, and there were no significant statistical differences between level Ⅱ and Ⅲ （z=- 1. 973,P= O. 052）. RT-PCR results showed that the relative expression of AR in breast cancer Ⅰ , Ⅱ and Ⅲ was 1.00±0.11, 0.53±0.15 and 0.59±0. 12, respectively, and there were significant statistical differences between level Ⅰ and Ⅱ（t= 6. 177, P = 0. 003）, Ⅰ and Ⅲ （t = 4. 162, P = 0. 014）, and there were no significant statistical differences between level Ⅱ and Ⅲ （t=- 1. 628,P=0. 179）. The protein and mRNA levels of AR in pathological grade I in breast cancer were significantly higher than that in grade II and II. The IOD ratio of AR protein in BT-549 and MDA-MB 231 was 1. 980±0. 101 and 0. 926±0. 072, respectively, and the latter was significantly lower than the former（t=14. 718,P〈0. 001）. The level of AR mRNA in MDA-MB-231 and BT-549 cells was 1. 000±0. 110 and 0. 020±0. 012, respectively, the level of AR mRNA in MDA-MB-231 cells was significantly lower than that of BT-549（t=107. 245,P〈0. 001）. The level of AR protein and miRNA in MDA-MB-231 cells were significantly lower than that of BT-549 cells. The data showed the levels of AR protein and mRNA in pathological grade I was much higher than those of Ⅱ and Ⅲ in breast cancers. RT-PCR showed the miR-873 and miR-185 in metastatic breast cancer cell lines was higher than in non-metastatic breast cancer cell lines, and the levels of miR 873 in grade I was significantly lower than that in grade Ⅱ and Ⅲ in breast cancers（F=11. 065,P=0. 010）. CONCLUSIONS The expression of AR is negatively correlated with histological grade and metastasis in breast cancer tissues and cells, the possible mechanism is associated with the up regulation of miR-873 in the high level breast cancer tissues, and then down regulates the expression of AR mRNA.