Department of Obstetrics and Gynecology, The Second Affiliated Hospital of Nantong University, Nantong, Jiangsu Province, China.
Department of Medical Laboratory, The First Affiliated Hospital of Xi'an Jiaotong University, China.
J Reprod Immunol. 2020 Aug;140:103129. doi: 10.1016/j.jri.2020.103129. Epub 2020 Apr 14.
The present study was meant for the discovery of the underlying functions of miR-485-5p in ovarian cancer concerning cisplatin resistance in vitro. RT-qPCR assessed the miR-485-5p expression in ovarian cancer cell lines, normal cells and cisplatin-resistant Cell line OVCA433-CR. After OVCA433-CR treated with 0,3,5umol/L cisplatin, miR-485-5p expressions were determined. MTT observed the cell cytotoxicity in OVCA433-CR after regulation of miR-485-5p. Targets can predicted the putative binding between miR-485-5p and PAK1 and Luciferase Assay verified this. RT-qPCR decided the inhibitory effect in between. MTT tested the cytotoxicity in different combinations of miR-485-5p and PAK1. Western Blot tested the phosphorylation of Pi3k and Akt in response to miR-485-5p and PAK1 interplay. We evaluated the role of Pi3k/Akt signaling in regulation of miR-485-5p and cisplatin resistance with Wortmannin. miR-485-5p was lower expressed in ovarian cancer cells than normal ones and even lower in OVCA433-CR than OVCA433. As the cisplatin concerntration increased, miR-485-5p decreased. miR-485-5p mimics induced lower cisplatin resistance while miR-485-5p inhibitor caused higher resistance. PAK1 targeted miR-485-5p and inhibited miR-485-5p. PAK1 inhibitor helped to lower the resistance to cisplatin caused by miR-485-5p upregulation. miR-485-5p mimics silenced Pi3k/Akt signaling and PAK1 inhibitor aggravated the silencing. Inhibition of Pi3k/Akt signaling increased miR-485-5p, thereby decreasing the cisplatin-resistance in OVCA433-CR. miR-485-5p decreased cisplatin resistance in ovarian cancer cells via Pi3k/Akt signaling, suggesting that miR-485-5p upregulation might alleviate the cisplatin resistance in ovarian patients.
本研究旨在发现 miR-485-5p 在体外顺铂耐药卵巢癌中的潜在作用。RT-qPCR 评估了卵巢癌细胞系、正常细胞和顺铂耐药细胞系 OVCA433-CR 中的 miR-485-5p 表达。用 0、3、5μmol/L 顺铂处理 OVCA433-CR 后,测定 miR-485-5p 的表达。MTT 观察 miR-485-5p 调节后 OVCA433-CR 的细胞毒性。预测 miR-485-5p 与 PAK1 之间的潜在结合,并用荧光素酶测定法验证。RT-qPCR 决定了两者之间的抑制作用。MTT 测试了 miR-485-5p 和 PAK1 不同组合的细胞毒性。Western Blot 检测了 miR-485-5p 和 PAK1 相互作用对 Pi3k 和 Akt 磷酸化的反应。我们用 Wortmannin 评估了 Pi3k/Akt 信号通路在调节 miR-485-5p 和顺铂耐药中的作用。miR-485-5p 在卵巢癌细胞中的表达低于正常细胞,在 OVCA433-CR 中的表达甚至低于 OVCA433。随着顺铂浓度的增加,miR-485-5p 减少。miR-485-5p 模拟物诱导的顺铂耐药性降低,而 miR-485-5p 抑制剂引起的耐药性增加。PAK1 靶向 miR-485-5p 并抑制 miR-485-5p。PAK1 抑制剂有助于降低 miR-485-5p 上调引起的顺铂耐药性。miR-485-5p 模拟物沉默 Pi3k/Akt 信号通路,PAK1 抑制剂加重沉默。抑制 Pi3k/Akt 信号通路增加了 miR-485-5p,从而降低了 OVCA433-CR 中的顺铂耐药性。miR-485-5p 通过 Pi3k/Akt 信号通路降低卵巢癌细胞的顺铂耐药性,表明 miR-485-5p 的上调可能减轻卵巢癌患者的顺铂耐药性。