白藜芦醇通过上调miR-361-3p逆转卵巢癌顺铂耐药的研究

Resveratrol reverses cisplatin resistance in ovarian cancer by up-regulating miR-361-3p

目的研究白藜芦醇(RES)逆转卵巢癌顺铂(DDP)耐药的作用机制。方法选取人卵巢癌SKOV3细胞,采用DDP构建SKOV3/DDP耐药细胞株。使用细胞计数试剂盒8(CCK-8)和5-乙炔基-2′脱氧尿嘧啶核苷(EdU)实验检测RES和DDP对SKOV3/DDP细胞的半数抑制浓度(IC_(50))和增殖能力的影响;采用实时荧光定量逆转录PCR(RT-qPCR)检测RES对微RNA-361-3p(miR-361-3p)的调控作用;采用流式细胞术检测RES和DDP对细胞凋亡的作用;细胞转染miR-361-3p抑制剂后检测DDP对细胞增殖和凋亡的作用。采用Western blot检测丝裂原活化蛋白激酶/细胞外信号调节激酶(MAPK/ERK)信号通路关键蛋白的表达。结果RES终浓度4μmol/L对SKOV3/DDP细胞生长无明显抑制作用(P>0.05),可作为最大安全浓度。RES+DDP组的IC_(50)明显低于DDP组(P<0.05)。RES可明显上调SKOV3/DDP细胞miR-361-3p的表达(P<0.05)。miR-361-3p抑制剂可以逆转DDP对SKOV3/DDP细胞的耐药性(P<0.05)。DDP组和RES+DDP组均可下调磷酸化ERK1/2(p-ERK1/2)、磷酸化P38(p-P38)蛋白水平(P<0.05),且RES+DDP组蛋白水平明显低于DDP组(P<0.05)。结论RES逆转SKOV3/DDP细胞对DDP的耐药可能与上调miR-361-3p和抑制MAPK/ERK信号通路的激活有关。

Objective To study the mechanism of resveratrol(RES)in reversing ovarian cancer cisplatin(DDP)-resistant.Methods Human ovarian cancer SKOV3 cells were selected,and DDP was used to construct SKOV3/DDP drug-resistant cell lines.The effects of RES and DDP on the half maximal inhibitory concentration(IC_(50))and proliferation ability of SKOV3/DDP cells were detected using the cell counting kit-8(CCK-8)and 5-ethynyl-2′-deoxyuridine(EdU)assay.The regulatory effect of RES on microRNA-361-3p(miR-361-3p)was detected by using real-time fluorescence quantitative reverse transcription PCR(RT-qPCR).Flow cytometry was used to detect the effects of RES and DDP on apoptosis.Additionally,the proliferation and apoptosis of DDP on cells were detected after transfecting the cells with a miR-361-3p inhibitor.Western blot was used to detect the expression of key proteins in the mitogen-activated protein kinase/extracellular signal-regulated kinase(MAPK/ERK)signaling pathway.Results The final concentration of RES at 4μmol/L had no significant inhibitory effect on the growth of SKOV3/DDP cells(P>0.05),which could be used as the maximum safe concentration.The IC_(50) of DDP in the RES+DDP group was significantly lower than that in the DDP group(P<0.05).RES significantly up-regulated the expression of miR-361-3p in SKOV3/DDP cells(P<0.05).The miR-361-3p inhibitor was able to reverse the resistance of DDP in SKOV3/DDP cells(P<0.05).Both the DDP group and RES+DDP group were able to down-regulate the protein levels of phosphorylated ERK1/2(p-ERK1/2)and phosphorylated P38(p-P38)(P<0.05).Additionally,the protein levels of p-ERK1/2 and p-P38 in the RES+DDP group were significantly lower than those in the DDP group(P<0.05).Conclusion The reversal of cisplatin resistance in SKOV3/DDP cells by RES may be related to the up-regulation of miR-361-3p and the inhibition of the activation of the MAPK/ERK signaling pathway.