hsa-miRNA27a和hsa-miRNA451通过调控MDRl/P-gp的表达和功能参与卵巢癌和乳腺癌细胞耐药

doi:10.3969/j.issn.1007-3969.2015.03.006

中国癌症杂志 ›› 2015, Vol. 25 ›› Issue (3): 190-198.doi: 10.3969/j.issn.1007-3969.2015.03.006

hsa-miRNA27a和hsa-miRNA451通过调控MDRl/P-gp的表达和功能参与卵巢癌和乳腺癌细胞耐药

李智敏¹，罗喜平¹，曾俐琴¹，彭秀红¹，王意¹，王泽华²

1. 广州医学院附属广东省妇幼保健院妇科，广东广州 510010 ；
2. 华中科技大学同济医学院附属协和医院妇产科，湖北武汉 430022

出版日期:2015-03-30 发布日期:2015-05-18
通信作者: 罗喜平　E-mail:lilyzhimin@sina.com

Expressions of miRNA27a and miRNA451 in ovarian cancer and breast cancer cells and its correlation with drug resistance by targeting MDR1/P-glycoprotein

LI Zhimin¹, LUO Xiping¹, ZENG Liqin¹, PENG Xiuhong¹, WANG Yi¹, WANG Zehua²

1.Department of Gynecology, Guangdong Women and Children Hospital, Guangzhou Guangdong 510010, China; 2.Department of Obstetrics and Gynecology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan Hubei 430022, China

Published:2015-03-30 Online:2015-05-18
Contact: LUO Xiping E-mail: lilyzhimin@sina.com

摘要/Abstract

摘要： 背景与目的：肿瘤细胞耐药是临床化疗失败的主要原因，微小RNA(microRNA，miRNA)在肿瘤细胞中的异常表达与耐药关系密切。本研究旨在探讨卵巢癌及乳腺癌细胞中hsa-miRNA27a和hsa-miRNA451的表达差异及其与耐药的关系。方法：用浓度递增法建立卵巢癌耐紫杉醇细胞系A2780/Taxol；颈环状引物实时定量聚合酶链反应(stem-loop quantitative real-time PCR，stem-loop RT-PCR)检测卵巢癌耐紫杉醇细胞A2780/Taxol和亲本细胞A2780以及乳腺癌耐阿霉素细胞MCF-7/ADM和亲本细胞MCF-7中hsa-miRNA27a和hsa-miRNA451的表达；利用Lipofectamine^TM 2000分别将成熟miRNA27a的模拟物、阻遏物及阴性对照(negative control，NC)RNA转染A2780和A2780/Taxol细胞，将成熟miRNA451的模拟物及NC转染MCF-7/ADM细胞；RT-PCR技术检测细胞MDR1 mRNA表达；蛋白[质]印迹法(Western blot)检测细胞中P-糖蛋白(P-glycoprotein，P-gp)的表达；采用四甲基偶氮唑蓝(MTT)法检测细胞增殖情况。结果：miRNA27a在A2780/Taxol细胞中高表达，与A2780细胞相比，表达增高2.2±0.30倍，差异有统计学意义(P<0.05)；miRNA451在MCF-7/ADM细胞中低表达，与MCF-7细胞相比，表达降低84%，差异有统计学意义(P<0.05)。A2780/Taxol细胞转染miRNA27a阻遏物后，MDR1 mRNA表达明显下降，与转染NC组相比，表达下降(39±0.14)%，差异有统计学意义(P<0.05)。P-gp相对表达量［(26±5.3)%)］与转染NC组的P-gp相对表达量［(43±6.7)%］比较，下降39%，差异有统计学意义(P<0.05)。对紫杉醇的敏感性增加，半数抑制浓度(IC₅₀)为0.53 μmol/L，与转染NC组IC50(6.8 μmol/L)相比，差异有统计学意义(P<0.05)。A2780细胞转染miRNA27a模拟物后，细胞的MDR1 mRNA表达升高，与转染NC组相比，升高(121±0.11)%，差异有统计学意义(P<0.05)；细胞对紫杉醇的敏感性下降，IC₅₀为0.2 μmol/L，与转染NC组IC₅₀(0.06 μmol/L)相比，差异有统计学意义(P<0.05)。MCF-7/ADM细胞转染miRNA451模拟物后，MDR1 mRNA表达明显下降，与转染NC组细胞相比，表达下降(65±12)%，差异有统计学意义(P<0.05)；P-gp相对表达量［(31±19)%)］与转染NC组细胞P-gp相对表达量［(83±12)%］相比，下降62%，差异有统计学意义(P<0.05)；对阿霉素的敏感性增加，IC₅₀为4.61 μmol/L，与转染NC组细胞IC₅₀(26 μmol/L)相比，差异有统计学意义(P<0.05)。结论：在卵巢癌耐紫杉醇细胞A2780/Taxol和乳腺癌耐阿霉素细胞MCF-7/ADM中，miRNA27a和miRNA451分别异常表达，它们可能分别通过间接或直接作用于MDR1/P-gp，参与肿瘤细胞耐药的发生、发展。

关键词: 卵巢癌, 乳腺癌, 紫杉醇, 阿霉素, 微小RNA, 耐药

Abstract: Background and purpose: Resistance of cancer cells to chemotherapy is a major clinical obstacle to successful treatment and leads to poor prognosis for the patients. MicroRNA (miRNA) plays a vital role in tumor cells response to chemotherapeutic agents. This study plans to investigate the expressions of miRNA27a and miRNA451 in ovarian cancer and breast cancer cells and its correlation with drug resistance. Methods: A2780/Taxol cells were established using stepwise selection; Stem-loop quantitative real-time PCR (stem-loop RT-PCR) was used to detect expression of miRNA27a and miRNA451 in ovarian cancer and breast cancer cells. The A2780 and A2780/Taxol cells were transfected with the mimics or inhibitors of miRNA27a or negative control (NC) RNA and the mimics of miRNA451 or NC were transfected into MCF-7/ADM cells by Lipofectamine^TM2000. The expression levels of MDR1 mRNA and P-glycoprotein (P-gp) were examined using RT-PCR and Western blot respectively. MTT method was used to analyze drug sensitivity. Results: The expression of miRNA27a was an average of (2.2±0.30) times higher in A2780/ Taxol cells than in A2780 cells, with a significant difference between the two groups (P<0.05). The expression of miRNA451 was lower by 84% in MCF-7/ADM cells than in MCF-7 cells, with a significant difference between the two groups (P<0.05). A2780/Taxol cells transfection with inhibitors of miRNA27a showed that the levels of MDR1 mRNA was decreased by (39±0.14)%, P-gp level [(26±5.3)%] decreased compared with the NC group [(43±6.7)%], the IC₅₀ (0.53 μmol/L) was less than the NC group (6.8 μmol/L), and there was a significant difference between two groups (P<0.05). Transfection of A2780 cells with mimics of miRNA27a led to increase MDR1 mRNA expression by (121±0.11)% and decrease the sensitivity to paclitaxel (IC₅₀: 0.2 μmol/L vs 0.06 μmol/L). There was a significant difference between two groups (P<0.05). Transfection of MCF-7/ADM cells with mimics of miRNA451 showed that expression of MDR1 mRNA was decreased by (65±12)%, P-gp [(31±19)%] was less than the NC group [(83±12)%], the sensitivity of cells to adriamycin enhanced and the IC₅₀ of adriamycin (4.61 μmol/L) was less than the NC group (26 μmol/L), and there was a significant difference between two groups (P<0.05). Conclusion: The expressions of miRNA27a and miRNA451 are deregulated in A2780/Taxol and MCF-7/ADM cells respectively, which may play vital roles in drug resistance by regulating MDR1/P-gp expression directly or indirectly.

Key words: Ovarian cancer, Breast cancer, Paclitaxel, Doxorubicin, MicroRNA, Drug resistance

李智敏,罗喜平,曾俐琴,等. hsa-miRNA27a和hsa-miRNA451通过调控MDRl/P-gp的表达和功能参与卵巢癌和乳腺癌细胞耐药[J]. 中国癌症杂志, 2015, 25(3): 190-198.

LI Zhimin, LUO Xiping, ZENG Liqin, et al. Expressions of miRNA27a and miRNA451 in ovarian cancer and breast cancer cells and its correlation with drug resistance by targeting MDR1/P-glycoprotein[J]. China Oncology, 2015, 25(3): 190-198.

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hsa-miRNA27a和hsa-miRNA451通过调控MDRl/P-gp的表达和功能参与卵巢癌和乳腺癌细胞耐药

Expressions of miRNA27a and miRNA451 in ovarian cancer and breast cancer cells and its correlation with drug resistance by targeting MDR1/P-glycoprotein

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