Zheng J, He J
Department of Gynecological Oncology, Sichuan Cancer Hospital, Sichuan Cancer Research Institute, Sichuan Cancer Prevention and Control Center, Sichuan Cancer Clinical Medical Research Center, University of Electronic Science and Technology of China Affiliated Cancer Hospital, Chengdu 610041, China.
Zhonghua Zhong Liu Za Zhi. 2025 Jun 23;47(6):498-507. doi: 10.3760/cma.j.cn112152-20240711-00285.
To explore the effect of miR-3591-3p targeting P53 on cisplatin resistance of ovarian cancer (OC) SKOV3/DDP cells. Target prediction and dual luciferase assay were used to validate miR-3591-3p targeting P53. SKOV3/DDP cells were divided into control group, DDP group, miR-3591-3p knockdown group (anti-miR-3591-3p), miR-3591-3p knockdown control group (anti-miR-NC), cisplatin+miR-3591-3p knockdown group (DDP+anti-miR-3591-3p), cisplatin+control group (DDP+anti-miR-NC), cisplatin+miR-3591-3p knockdown+P53 knockdown group (DDP+anti-miR-3591-3p+sh-P53), cisplatin+miR-3591-3p knockdown+P53 knockdown control group (DDP+anti-miR-3591-3p+sh-NC). CCK-8 assay was used to detect cell survival rate and IC value; Flow cytometry were used to detect cell cycle and apoptosis; RT-qPCR was used to detect miR-3591-3p and P53 mRNA levels in cells; Western blot was used to detect P53, P-gp, MRP1, Ki-67, CyclinD1, Bcl-2, and Bax protein levels in cells. Nude mice were divided into control group, DDP group, miR-3591-3p knockdown group (anti-miR-3591-3p), miR-3591-3p knockdown control group (anti-miR-NC), and cisplatin+miR-3591-3p knockdown group (DDP+anti-miR-3591-3p). Subcutaneous injection of SKOV3/DDP cells was used to prepare transplanted tumor model. Tumor volume, mass, miR-3591-3p, P53 mRNA and protein levels in tumor tissue were detected. MiR-3591-3p and P53 had binding sites. After overexpression of miR-3591-3p, wild-type P53 luciferase activity was decreased (＜0.05); However, there was no significant difference in the luciferase activity of mutant P53 (＞0.05). After DDP treatment or knockdown of miR-3591-3p expression, miR-3591-3p level in cells was decreased, the mRNA and protein levels of P53 were increased; the cell survival rate and IC value were decreased [DDP group 24, 36, 48 h IC (21.26±2.95)mg/L,(17.38±1.93)mg/L and (13.76±1.46)mg/L, control group (41.06±4.39)mg/L, (36.15±3.46)mg/L and(29.87±1.39)mg/L; anti-miR-3591-3p group 24,36,48 h IC (19.96±2.19)mg/L, (17.62±3.52)mg/L and (13.05±1.53)mg/L,anti-miR-NC group (43.37±3.83)mg/L, (40.47±2.82)mg/L and (31.41±0.73)mg/L], the proportion of S phase was decreased, the proportion of G/G phase and cell apoptosis rate [DDP group (27.00±2.00)%, Control group (3.33±1.53)%; anti-miR-3591-3p group (28.98±3.14)%, anti-miR-NC group (4.05±1.96)%] were increased; P-gp, MRP1, Ki-67, CyclinD1, Bcl-2 protein levels were decreased, while Bax protein level was increased (all ＜0.05). Knocking down miR-3591-3p could enhance the impact of DDP on the above indicators (all ＜0.05). Knocking down P53 expression could inhibit the impact of miR-3591-3p deletion on the above indicators (all ＜0.05). After DDP treatment or knockdown of miR-3591-3p, the tumor volume and weight of nude mice were decreased [DDP group 14,21,28 d volume (284.26±24.51)mm,(563.21±44.17)mm and (741.32±72.01)mm,control group (384.25±41.25)mm, (840.32±71.27)mm and (1 242.47±100.54)mm; anti-miR-3591-3p group 14,21,28 d volume (274.47±27.77)mm, (584.68±61.14)mm and (815.24±73.19)mm, anti-miR-NC group (355.47±46.84)mm, (804.24±79.54)mm and (1 350.47±108.37)mm; DDP group weight (0.85±0.15)g,control group (1.34±0.12)g; anti-miR-3591-3p group (0.88±0.14)g, anti-miR-NC group (1.34±0.10)g],miR-3591-3p level in tumor tissue was decreased, and the mRNA and protein levels of P53 were increased (all ＜0.05); Knocking down miR-3591-3p could enhance the effect of DDP on the above indicators (all ＜0.05). MiR-3591-3p targeting P53 enhances cisplatin resistance in SKOV3/DDP cells.
探讨miR-3591-3p靶向P53对卵巢癌（OC）SKOV3/DDP细胞顺铂耐药性的影响。采用靶标预测和双荧光素酶报告基因检测法验证miR-3591-3p靶向P53。将SKOV3/DDP细胞分为对照组、顺铂组、miR-3591-3p敲低组（抗-miR-3591-3p）、miR-3591-3p敲低对照组（抗-miR-NC）、顺铂+miR-3591-3p敲低组（顺铂+抗-miR-3591-3p）、顺铂+对照组（顺铂+抗-miR-NC）、顺铂+miR-3591-3p敲低+P53敲低组（顺铂+抗-miR-3591-3p+sh-P53）、顺铂+miR-3591-3p敲低+P53敲低对照组（顺铂+抗-miR-3591-3p+sh-NC）。采用CCK-8法检测细胞存活率和IC值；采用流式细胞术检测细胞周期和凋亡；采用RT-qPCR法检测细胞中miR-3591-3p和P53 mRNA水平；采用蛋白质免疫印迹法检测细胞中P53、P-糖蛋白（P-gp）、多药耐药相关蛋白1（MRP1）、细胞增殖抗原Ki-67、细胞周期蛋白D1（CyclinD1）、凋亡抑制蛋白Bcl-2和促凋亡蛋白Bax的蛋白水平。将裸鼠分为对照组、顺铂组、miR-3591-3p敲低组（抗-miR-3591-3p）、miR-3591-3p敲低对照组（抗-miR-NC）和顺铂+miR-3591-3p敲低组（顺铂+抗-miR-3591-3p）。皮下注射SKOV3/DDP细胞制备移植瘤模型。检测肿瘤体积、质量、肿瘤组织中miR-3591-3p、P53 mRNA和蛋白水平。miR-3591-3p与P53存在结合位点。miR-3591-3p过表达后，野生型P53荧光素酶活性降低（＜0.05）；而突变型P53荧光素酶活性无显著差异（＞0.05）。顺铂处理或敲低miR-3591-3p表达后，细胞中miR-3591-3p水平降低，P53的mRNA和蛋白水平升高；细胞存活率和IC值降低[顺铂组24、36、48 h的IC值分别为（21.26±2.95）mg/L、（17.38±1.93）mg/L和（13.76±1.46）mg/L，对照组分别为（41.06±4.39）mg/L、（36.15±3.46）mg/L和（29.87±1.39）mg/L；抗-miR-3591-3p组24、36、48 h的IC值分别为（19.96±2.19）mg/L、（17.62±3.52）mg/L和（13.05±1.53）mg/L，抗-miR-NC组分别为（43.37±3.83）mg/L、（40.47±2.82）mg/L和（31.41±0.73）mg/L]，S期比例降低，G0/G1期比例和细胞凋亡率升高[顺铂组为（27.00±2.00）%，对照组为（3.33±1.53）%；抗-miR-3591-3p组为（28.98±3.14）%，抗-miR-NC组为（4.05±1.96）%]；P-gp、MRP1、Ki-67、CyclinD1、Bcl-2蛋白水平降低，而Bax蛋白水平升高（均＜0.05）。敲低miR-3591-3p可增强顺铂对上述指标的影响（均＜0.0
