丹皮酚通过下调miR-21并抑制PI3K/AKT通路抑制Hela细胞侵袭、转移及增殖

Paeonol inhibits the invasion,metastasis and proliferation of Hela cells by down-regulating miR-21 and inhibiting PI3K/AKT pathway

目的:探讨丹皮酚对Hela细胞增殖、侵袭及迁移的影响及可能机制。方法:使用不同浓度(50、100、200和400 mg/L)丹皮酚处理Hela细胞株,分为正常对照组(NC组)、不同浓度丹皮酚组、miR-NC组、miR-21组、丹皮酚+miR-NC和丹皮酚+miR-21组。PI3K的激动剂740Y-P(25μg/mL)加入丹皮酚组、NC组,分别定义为丹皮酚+740Y-P组、740Y-P组。MTT检测细胞增殖能力;Transwell实验检测细胞侵袭、迁移能力;实时荧光定量PCR检测细胞miR-21表达水平;Western blot检测细胞PI3K/AKT信号通路相关蛋白、基质金属蛋白酶2(matrix metalloproteinase 2,MMP2)、MMP9水平。结果:丹皮酚可抑制Hela细胞增殖能力(P<0.05),抑制作用与浓度呈正相关。选择200 mg/L的丹皮酚进行后续研究。与NC组比较,丹皮酚组侵袭及迁移细胞数量、MMP2蛋白、MMP9蛋白、miR-21表达水平明显降低(P<0.01)。与NC组比较,丹皮酚组细胞凋亡率增加,G_(1)期延长,S期、G_(2)期缩短(P<0.01)。与NC组、miR-NC组相比,48、72 h时miR-21组细胞活力明显升高(P<0.05),克隆、侵袭及迁移细胞数量明显升高(P<0.05)。与丹皮酚组、丹皮酚+miR-NC组比较,丹皮酚+miR-21组克隆、侵袭及迁移细胞数升高(P<0.05)。与NC组比较,丹皮酚组细胞p-PI3K、p-AKT蛋白表达水平明显下降(P<0.01);与丹皮酚+miR-NC组比较,丹皮酚+miR-21组细胞p-PI3K、p-AKT蛋白表达水平明显升高(P<0.01)。与丹皮酚组比较,丹皮酚+740Y-P组克隆、侵袭及迁移细胞数升高(P<0.01),与740Y-P组比较,丹皮酚+740Y-P组克隆、侵袭及迁移细胞数降低(P<0.01)。结论:丹皮酚可下调miR-21,抑制PI3K/AKT通路,进而抑制宫颈癌细胞的增殖、侵袭及迁移。

Objective:To investigate the effect of paeonol on proliferation,invasion and migration of Hela cells and its possible mechanism.Methods:Hela cells were treated with different concentrations of paeonol(50,100,200 and 400 mg/L)and divided into normal control group(NC group),paeonol group,miR-NC group,miR-21 group,paeonol+miR-NC and paeonol+miR-21 groups.The agonist 740Y-P(25μg/mL)of PI3K was added to paeonol group and NC group,which were defined as paeonol+740Y-P group and 740Y-P group respectively.Cell proliferation was detected by MTT assay.Transwell assay was used to detect cell invasion and migration.The expression of miR-21 was detected by real-time PCR.The protein levels of PI3K/AKT signaling pathway were detected by Western blot.Results:Paeonol could inhibit the proliferation of Hela cells(P<0.05).200 mg/L paeonol was selected for follow-up study.Compared with NC group,the number of invasive and migratory cells and the expression level of miR-21,MMP2 and MMP9 in paeonol group were significantly decreased(P<0.01).Compared with NC group,the apoptosis rate of paeonol group was increased,G_(1) phase was prolonged,S phase and G_(2) phase were shortened(P<0.01).Compared with NC group and miR-NC group,the cell viability of miR-21 group increased significantly at 48 h and 72 h(P<0.05),and the number of clone,invasion and migration cells increased significantly(P<0.05).Compared with paeonol group and paeonol+miR-NC group,the number of clone,invasion and migration cells in paeonol+miR-21 group increased(P<0.05).Compared with NC group,the expression levels of p-PI3K and p-AKT in paeonol group were significantly decreased(P<0.01).Compared with paeonol+miR-NC group,the expression levels of p-PI3K and p-AKT in paeonol+miR-21 group were significantly increased(P<0.01).Compared with paeonol group,the number of clonal,invasive and migratory cells in paeonol+740Y-P group increased(P<0.01),and the number of clonal,invasive and migratory cells in paeonol+740Y-P group decreased(P<0.01).Conclusion:Paeonol can down-regulate miR-21,inhibit PI3K/AKT pathway,and inhibit the proliferation,invasion and migration of cervical cancer cells.