Chen Yanyan, Wang Li, Zhou Jiefang
Department of Outpatient Pharmacy, Ningbo Women & Children's Hospital, Ningbo, China.
Department of Clinical Pharmacology, Shaoxing Traditional Chinese Medicine Hospital, Shaoxing, China.
J Obstet Gynaecol Res. 2019 Nov;45(11):2243-2254. doi: 10.1111/jog.14079. Epub 2019 Aug 14.
Efficacy of platinum based-chemotherapy is limited by cisplatin (DDP) resistance, however, the underlying mechanism of cisplatin resistance remains unclear. We aimed to investigate the role of miR-1271 in cisplatin-resistant ovarian cancer cells.
Transfection of miR-1271 mimic and inhibitor was performed to study the role of miR-1271 in ovarian cancer. Cell viability was assessed by Cell Counting Kit (CCK)-8 assay. Flow cytometry was used to determine the apoptosis rates. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blot were used to detect mRNA and protein expressions. Target predicted by Targetscan7.2 was confirmed by dual-luciferase activity assay. Mammalian target of rapamycin (mTOR) siRNA (simTOR) co-transfection was performed to verify the role of mTOR in the suppression effect of miR-1271 on ovarian cancer.
In SKOV3 cells, miR-1271 overexpression significantly decreased cell viability and up-regulated apoptosis rate (from 5.54% of control to 24.03%). MiR-1271 adversely affected SKOV3 cell migration and invasion, and induced the upregulation of E-cadherin and downregulation of N-cadherin and alpha-smooth muscle actin (α-SMA). Moreover, SKOV3/DDP cells had a lower miR-1271 level, and enhancing miR-1271 contributed strongly to cisplatin-induced apoptosis through altering the expressions of B-cell lymphoma-2 associated X protein (BAX), cleaved caspase-3 and B-cell lymphoma 2 (Bcl-2). In contrast, the opposite result was observed in miR-1271 inhibitor. mTOR was identified to be a target of miR-1271. SimTOR partially reversed the increased cell viability under the effect of miR-1271 inhibitor.
Our data indicate that miR-1271 can inhibit the ovarian cancer epithelial-mesenchymal transition (EMT) and sensitize resistant cells to cisplatin-induced apoptosis through blocking mTOR expression.
铂类化疗的疗效受顺铂（DDP）耐药性限制，然而，顺铂耐药的潜在机制仍不清楚。我们旨在研究miR-1271在顺铂耐药卵巢癌细胞中的作用。
进行miR-1271模拟物和抑制剂转染以研究miR-1271在卵巢癌中的作用。通过细胞计数试剂盒（CCK）-8法评估细胞活力。流式细胞术用于测定凋亡率。定量逆转录聚合酶链反应（qRT-PCR）和蛋白质免疫印迹法用于检测mRNA和蛋白质表达。通过双荧光素酶活性测定法证实Targetscan7.2预测的靶标。进行雷帕霉素哺乳动物靶蛋白（mTOR）小干扰RNA（simTOR）共转染以验证mTOR在miR-1271对卵巢癌的抑制作用中的作用。
在SKOV3细胞中，miR-1271过表达显著降低细胞活力并上调凋亡率（从对照的5.54%升至24.03%）。miR-1271对SKOV3细胞迁移和侵袭产生不利影响，并诱导E-钙黏蛋白上调以及N-钙黏蛋白和α-平滑肌肌动蛋白（α-SMA）下调。此外，SKOV3/DDP细胞的miR-1271水平较低，增强miR-1271通过改变B细胞淋巴瘤-2相关X蛋白（BAX）、裂解的半胱天冬酶-3和B细胞淋巴瘤2（Bcl-2）的表达，对顺铂诱导的凋亡有强烈促进作用。相反，在miR-1271抑制剂组中观察到相反的结果。mTOR被确定为miR-1271的靶标。SimTOR部分逆转了miR-1271抑制剂作用下细胞活力的增加。
我们的数据表明，miR-1271可抑制卵巢癌上皮-间质转化（EMT），并通过阻断mTOR表达使耐药细胞对顺铂诱导的凋亡敏感。
