微小RNA-122在人乳腺癌组织中的表达及对体外培养人乳腺癌细胞他莫昔芬耐药的影响

Expression of microRNA-122 in human breast cancer tissue and the influence on tamoxifen resistance of human breast cancer cells cultured in vitro

目的探讨微小RNA-122(miR-122)在人乳腺癌组织中的表达及对体外培养人乳腺癌细胞他莫昔芬耐药的影响。方法收集2015年1-6月河北省保定市第二中心医院行乳腺切除术的56例Luminal型乳腺癌患者的肿瘤组织及癌旁组织标本,采用实时荧光定量聚合酶链反应(q PCR)法检测miR-122表达量。所有患者规范口服他莫昔芬3年,按照实体瘤疗效评价标准划分为他莫昔芬敏感者和他莫昔芬耐药者。体外培养人乳腺癌细胞株MCF-7,他莫昔芬长期浓度梯度递增法体外建立MCF-7TamR耐药细胞株;转染miR-122抑制剂(抑制剂组),采用噻唑蓝(MTT)法检测他莫昔芬对MCF-7细胞、MCF-7TamR细胞以及抑制剂组MCF-7TamR细胞增殖活性的影响。采用q PCR法检测雌激素受体α(ER-α)、人类表皮生长因子受体2(HER-2)、丝裂原活化蛋白激酶3(MAPK3) mRNA表达。采用蛋白质印迹法检测ER-α、HER-2、细胞外信号调节激酶/丝裂原活化蛋白激酶(ERK/MAPK)、磷酸化ERK-MAPK(p-ERK/MAPK)蛋白表达。结果乳腺癌组织中miR-122相对表达量明显高于癌旁组织[(0. 70±0. 38)比(0. 24±0. 10)],他莫昔芬耐药患者(11例)肿瘤组织中miR-122相对表达量明显高于敏感患者(45例)[(0. 89±0. 13)比(0. 67±0. 15)](均P <0. 001)。体外成功建立MCF-7TamR耐药细胞株,不同浓度他莫昔芬(0. 1、0. 5、2. 5、12. 5、62. 5、312. 5μmol/L)对MCF-7TamR细胞的增殖抑制率分别均明显低于MCF-7细胞(均P <0. 05);MCF-7TamR细胞的耐药指数为4. 97。转染miR-122抑制剂后,抑制剂组MCF-7TamR细胞miR-122表达量为(0. 14±0. 04),明显低于空白对照组[CTL组:(0. 67±0. 09)]和空转染组[NC组:(0. 65±0. 08)](均P <0. 001)。不同浓度他莫昔芬对抑制剂组MCF-7TamR细胞的增殖抑制率明显高于CTL组和NC组(均P <0. 05)。经q PCR法,抑制剂组MCF-7TamR细胞ER-αmRNA表达量为(1. 02±0. 17),明显高于CTL组和NC组;HER-2和MAPK3 mRNA表达量为(0. 37±0. 09)和(0. 25±0. 07),明显低于CTL组和NC组(均P <0. 05)。经蛋白质印迹法,抑制剂组MCF-7TamR细胞ER-α蛋白表达量为(0. 91±0. 18),明显高于CTL组和NC组;HER-2和p-ERK/MAPK蛋白表达量为(0. 24±0. 09)和(1. 15±0. 26),明显低于CTL组和NC组(均P <0. 05)。结论乳腺癌组织中miR-122表达增高,下调miR-122可明显逆转乳腺癌MCF-7TamR耐药细胞对他莫昔芬的抗性,并抑制MCF-7TamR细胞增殖,其机制可能与阻断ERK/MAPK信号通路,提高ER表达有关。

Objective To investigate the expression of microRNA-122( miR-122) in human breast cancer tissue and its effect on tamoxifen( Tam) resistance of human breast cancer cells cultured in vitro. Methods Tumor tissue and adjacent normal tissue were separated from 56 patients with Luminal breast cancer who had mastectomy in the Second Central Hospital of Baoding City,Hebei Province from January to June 2015. Real-time fluorescence quantitative polymerase chain reaction( q PCR) was used to detect miR-122 expression. All patients took Tam for3 years;Tam-resistance was determined according to the Response Evaluation Criteria in Solid Tumors. Breast cancer cell line MCF-7 was cultured in vitro and Tam-resistance was initiated by stepwise revulsion with Tam.Tam-resistance cell line MCF-7 TamR was transfected by miR-122 inhibitor( inhibitor group). Cell proliferation was detected by methyl thiazolyl tetrazolium( MTT) assay. Estrogen receptor-α( ER-α),human epithelial growth factor receptor-2( HER-2),mitogen-activated protein kinase 3( MAPK3) mRNA expressions were detected by q PCR.ER-α,HER-2,extracellular signal-regulated kinase/mitogen-activated protein kinase( ERK/MAPK) and phosphoERK/MARK( p-ERK/MAPK) expressions were detected by western blotting. Results Expression level of miR-122 in tumor tissue was higher than that in normal tissue [( 0. 70 ± 0. 38) vs( 0. 24 ± 0. 10) ];expression level of miR-122 in 11 patients with Tam-resistance was higher than that in 45 patients with Tam-sensitivity[( 0. 89 ± 0. 13) vs( 0. 67 ± 0. 15) ]( both P < 0. 001). Proliferation inhibition rates induced by 0. 1,0. 5,2. 5,12. 5,62. 5 and 312. 5 μmol/L Tam of MCF-7 TamRcells were significantly lower than those of MCF-7 cells( all P <0. 05);Tam-resistance index was 4. 97. Expression level of miR-122 in MCF-7 TamRcells in inhibitor group was( 0. 14 ± 0. 04),which was significantly lower than that in blank control group[CTL group:( 0. 67 ± 0. 09) ]and null transfection group[NC group:( 0. 65 ± 0. 08) ]( both P < 0. 05). Proliferation inhibition rates induced by different concentrations of Tam of MCF-7 TamRcells in inhibitor group were significantly higher than those in CTL group and NC group( all P < 0. 001). q PCR showed that ER-α mRNA expression in inhibitor group( 1. 02 ± 0. 17)was higher and HER-2 and MAPK3 mRNA expressions[( 0. 37 ± 0. 09),( 0. 25 ± 0. 07) ] were lower than those in CTL group and NC group( all P < 0. 05). Western blotting showed that ER-α expression( 0. 91 ± 0. 18) was higher and HER-2 and p-ERK/MAPK expressions [( 0. 24 ± 0. 09),( 1. 15 ± 0. 26) ] were lower than those in CTL group and NC group( all P < 0. 05). Conclusions Expression of miR-122 increases in breast cancer tissue.Down-regulation of miR-122 can reverse the resistance of breast cancer cells MCF-7 TamRto tamoxifen and inhibit cell proliferation,which may be related to blocking ERK/MAPK signaling pathway and increasing ER sensitivity.