川楝素调控miR-409-3p对前列腺癌细胞增殖、迁移、侵袭及VEGF/VEGFR2通路的影响

The effect of toosendanin on the proliferation,migration,invasion and vascular endothelial growth factor/vascular endothelial growth factor receptor 2 pathway of prostate cancer cells by regulating microRNA-409-3p

目的探讨川楝素(TSN)调控微小RNA-409-3p(miR-409-3p)对前列腺癌细胞增殖、迁移、侵袭及血管内皮生长因子(VEGF)/血管内皮生长因子受体2(VEGFR2)通路的影响。方法使用0、10、20、40、80和160μmol/L的TSN处理PC3细胞,采用噻唑蓝法检测PC3细胞存活率,选择最佳药物浓度。将细胞分为对照组(Control组)、TSN低浓度组(TSN-L组)、TSN中浓度组(TSN-M组)、TSN高浓度组(TSN-H组)、TSN高浓度+miR-409-3p拮抗剂阴性对照组(TSN-H+antagomiR-NC组)、TSN高浓度+miR-409-3p拮抗剂组(TSN-H+antagomiR-409-3p组)。采用实时荧光定量聚合酶链反应(qRT-PCR)检测PC3细胞中miR-409-3p表达;EdU法检测PC3细胞增殖水平;Transwell小室实验和划痕愈合实验检测PC3细胞的侵袭和迁移能力;Western blot检测细胞周期蛋白D1(CyclinDl)、细胞周期蛋白依赖性激酶4(CDK4)、VEGF、VEGFR2蛋白表达。结果与0μmol/L比较,10、20、40、80和160μmol/L的TSN细胞存活率显著降低(P<0.05),选择20、40、80μmol/L的TSN用于后续实验。与Control组比较,TSN-L组、TSN-M组和TSN-H组PC3细胞EdU阳性率、细胞侵袭数、划痕愈合率、CyclinDl、CDK4、VEGF、VEGFR2蛋白表达显著降低(P<0.05),miR-409-3p表达显著增加(P<0.05),且呈浓度依赖性。与TSN-H+antagomiR-NC组比较,TSN-H+antagomiR-409-3p组PC3细胞EdU阳性率、细胞侵袭数、划痕愈合率、CyclinDl、CDK4、VEGF、VEGFR2蛋白表达显著增加(P<0.05),miR-409-3p表达显著降低(P<0.05)。结论TSN通过上调miR-409-3p表达抑制VEGF/VEGFR2通路的激活,进而抑制PC3细胞增殖、迁移、侵袭。

Objective To investigate the effects of toosendanin(TSN)on the proliferation,migration,invasion and vascular endothelial growth factor/vascular endothelial growth factor receptor 2(VEGF/VEGFR2)pathway of prostate cancer cells by regulating microRNA-409-3 p(miR-409-3 p).Methods PC3 cells were treated with TSN of 0,10,20,40,80,and 160μmol/L,and the survival rate of PC3 cells was detected by thiazole blue method,and the optimal drug concentration was selected.The cells were divided into control group,low concentrations toosendanin group(TSN-L group),medium concentrations toosendanin group(TSN-M group),high concentrations toosendanin group(TSN-H group),high concentration TSN+miR-409-3 p antagonist negative control group(TSN-H+antagomiR-NC group),high concentration TSN+miR-409-3 p antagonist group(TSN-H+antagomiR-409-3 p group).Real-time quantitative fluorescent polymerase chain reaction(qRT-PCR)was used to detect the expression of miR-409-3 p in PC3 cells;EdU method was used to detect the proliferation level of PC3 cells;Transwell chamber experiment and scratch healing experiment were used to detect the abilities of invasion and migration in PC3 cells;Western blot was used to detect the expression of CyclinDl,cyclin dependent kinase 4(CDK4),VEGF,and VEGFR2 proteins.Results Compared with 0μmol/L,the survival rate of PC3 cells treated with 10,20,40,80 and 160μmol/L TSN was significantly reduced(P<0.05),20,40,80μmol/L TSN were selected for subsequent experiments.Compared with the control group,the EdU positive rate,number of cell invasion,scratch healing rate,the expressions of CyclinDl,CDK4,VEGF,and VEGFR2 protein of PC3 cells in the TSN-L group,TSN-M group,and TSN-H group were significantly reduced(P<0.05),the expressions of miR-409-3 p were significantly increased(P<0.05),with a concentration dependent manner.Compared with the TSN-H+antagomiR-NC group,the EdU positive rate,number of cell invasion,scratch healing rate,the expressions of CyclinDl,CDK4,VEGF,and VEGFR2 protein of PC3 cells in the TSN-H+antagomiR-409-3 p group were significantly increased(P<0.05),the expressions of miR-409-3 p were significantly reduced(P<0.05).Conclusions TSN inhibits the activation of the VEGF/VEGFR2 pathway by up-regulating the expression of miR-409-3 p,thereby inhibiting the proliferation,migration,and invasion of PC3.