摘要
目的研究微小RNA27a（miR-27a）在卵巢上皮性癌（卵巢癌）紫杉醇耐药细胞中的表达及其与耐药的关系。方法采用实时PCR技术检测卵巢癌A2780细胞及其紫杉醇耐药细胞A2780／Taxol中miR-27a的表达水平。利用脂质体lipofectamine2000将成熟miR-27a的模拟物、阻遏物及阴性对照（NC）转染A2780和A2780／Taxol细胞，实时PCR技术检测转染细胞中MDR1基因mRNA的表达；蛋白印迹法检测转染细胞中P一糖蛋白（P—gp）和同源结构域相关的蛋白激酶2（HIPK2）蛋白的表达；采用四甲基偶氮唑蓝比色法、流式细胞仪分别检测紫杉醇作用于转染后细胞的增殖情况及细胞凋亡率。结果（1）miR-27a在A2780／Taxol细胞中高表达，与A2780细胞相比表达水平增高2．2倍，差异有统计学意义（P〈0．05）。（2）转染miR-27a阻遏物后，A2780／Taxol细胞中MDRlmRNA的表达水平为0．61±0．14，与转染NC细胞（表达水平为1．00）比较下降39％，差异有统计学意义（P〈0．05）；P．gP蛋白的表达水平为（26±5）％，HIPK2蛋白的表达水平为（30±6）％，分别与转染NC细胞的P-gp蛋白[（43±7）％]和HIPK2蛋白[（19±4）％]的表达水平比较，差异均有统计学意义（P〈0．05）；A2780／Taxol细胞对紫杉醇的敏感性增加，半数抑制浓度（IC50）为0．5μmo]／L，与转染Nc细胞（Ic。为6．8Iμmol／L）比较，差异有统计学意义（P〈0．05）；细胞凋亡率明显升高，达（32．5±3．6）％，与转染NC细胞的凋亡率（5．6±2．1）％比较，差异有统计学意义（P〈0．05）。（3）转染miR-27a模拟物后，A2780细胞中MDR1 mRNA的表达水平为2．21±0．11，与转染NC细胞（表达水平为1．00）比较升高121％，差异有统计学意义（P〈0．05）。结论miR-27a在卵巢癌紫杉醇耐药细胞中高表达，其可能通过调控靶基因HIPK2，间接调节MDR1及P—gP的表达和功能而参与耐药。
Objective To investigate the expression of microRNA 27a （miR-27a） and relationship with drug resistance in human ovarian cancer A2780/Taxol cells. Methods A stern-loop-mediated real-time PCR was used to detect miR-27a expression in A2780 and A2780/Taxol cells. The ceils were transfected with the mimics or inhibitors of miR-27a or negative control RNA （ NC ） by lipofectamine 2000. The expressions of MDR1 gene, P-glycoprotein （P-gp） and homeodomain-interacting protein kinase 2 （HIPK2） protein levels were measured by real-time PCR and western blot respectively. Methyl thiazolyl tetrazolium （MTT） assay was used to analyze drug sensitivity. Apoptosis analysis was measured by fluorescence activated cell sorter （FACS）. Results （ 1 ） miR-27a was an average of 2.2-fold higher expression level in A2780/Taxol cells than that in A2780 cells, with a significant difference between the two groups （ P 〈 0. 05 ）. （2） A2780/Taxol cells transfection with inhibitors of miR-27a showed that the levels of MDR1 mRNA was decreased by 39%, P-gp protein level [ （26 ± 5） % ] decreased than that in the NC group [ （43 ± 7） % ], HIPK2 protein level [ （30 ±6）% ] increased than that in the NC group [ （ 19 ±4）% ], the 50% inhibition concentration （0. 5 μmol/L） was less than that in the NC group （6. 8 μmol/L）, apoptosis rate [ （32. 5 ± 3.6） % ] was higher than that in the NC group [ （5.6 ± 2. 1 ） % ], and there were significant differences between two groups （all P 〈0. 05 ）. （3） Transfection of A2780 cells with mimics of miR-27a led to increase MDRI mRNA expression by 121% as compared with one transfection with NC （P 〈 0. 05 ）. Conclusion The expression of miR-27a is upregulated in A2780/Taxol ceils, which may regulate MDR1 and P-gp expression by targeting HIPK2.
出处
《中华妇产科杂志》
CAS
CSCD
北大核心
2010年第5期372-375,共4页
Chinese Journal of Obstetrics and Gynecology
基金
国家自然科学基金（30901585）
关键词
卵巢肿瘤
紫杉酚
微RNAS
抗药性
肿瘤
Ovarian neoplasms
Paclitaxel
MicroRNAs
Drug resistance, neoplasm
