[The effect of miR-3591-3p targeting P53 on cisplatin resistance in ovarian cancer SKOV3/DDP cells]

Abstract

Objective: To explore the effect of miR-3591-3p targeting P53 on cisplatin resistance of ovarian cancer (OC) SKOV3/DDP cells. Methods: 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 IC₅₀ 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. Results: MiR-3591-3p and P53 had binding sites. After overexpression of miR-3591-3p, wild-type P53 luciferase activity was decreased (P＜0.05); However, there was no significant difference in the luciferase activity of mutant P53 (P＞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 IC₅₀ value were decreased [DDP group 24, 36, 48 h IC₅₀ (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 IC₅₀ (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 G₀/G₁ 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 P＜0.05). Knocking down miR-3591-3p could enhance the impact of DDP on the above indicators (all P＜0.05). Knocking down P53 expression could inhibit the impact of miR-3591-3p deletion on the above indicators (all P＜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)mm³,(563.21±44.17)mm³ and (741.32±72.01)mm³,control group (384.25±41.25)mm³, (840.32±71.27)mm³ and (1 242.47±100.54)mm³; anti-miR-3591-3p group 14,21,28 d volume (274.47±27.77)mm³, (584.68±61.14)mm³ and (815.24±73.19)mm³, anti-miR-NC group (355.47±46.84)mm³, (804.24±79.54)mm³ and (1 350.47±108.37)mm³; 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 P＜0.05); Knocking down miR-3591-3p could enhance the effect of DDP on the above indicators (all P＜0.05). Conclusion: MiR-3591-3p targeting P53 enhances cisplatin resistance in SKOV3/DDP cells.