miR-520a-5p通过调控有氧糖酵解诱导前列腺癌细胞多西他赛耐药

miR-520a-5p induces docetaxel resistance in prostate cancer cells by regulating aerobic glycolysis

摘要:

目的 探索miR-520a-5p对前列腺癌增殖能力和多西他赛耐药性的影响及其作用机制。方法 使用RT-qPCR检测PC-3/DU145前列腺癌细胞及RWPE-1前列腺正常上皮细胞中miR-520a-5p表达水平；使用免疫印迹检测miR-520a-5p是否调控糖酵解关键酶己糖激酶2（hexokinase 2，HK2）和磷酸果糖激酶（phosphofructokinase，PFKM）表达水平；使用CCK-8实验检测miR-520a-5p对前列腺癌细胞增殖能力和多西他赛耐药性的影响；使用ELISA评估培养上清中葡萄糖消耗水平和乳酸生成水平。依据转染物的不同，将所有细胞分成4组：minic组、minic NC组、inhibitor组、inhibitor NC组。结果 miR-520a-5p在前列腺癌细胞中表达下调（P<0.001）。与minic NC组比较，minic组可显著降低PC-3和DU145前列腺癌细胞的增殖能力（P<0.001）、多西他赛半抑制浓度（IC₅₀）、葡萄糖消耗水平和乳酸生成水平（P<0.001）；与inhibitor NC组比较，inhibitor组可明显增强PC-3和DU145前列腺癌细胞的增殖能力（P<0.001）、葡萄糖消耗水平和乳酸生成水平（P<0.001）。Starbase数据库提示miR-520a-5p与HK2和PFKM存在结合位点；免疫印迹结果显示，miR-520a-5p对HK2和PFKM的表达存在负调控关系。使用2-DG抑制前列腺癌细胞糖酵解可恢复miR-520a-5p对前列腺癌增殖能力的影响。结论 前列腺癌中miR-520a-5p可能通过上调糖酵解关键酶HK2和PFKM表达水平增强前列腺癌细胞有氧糖酵解诱导多西他赛化疗耐药。

Abstract:

Objective To explore the effects of miR-520a-5p on the proliferation and docetaxel resistance of prostate cancer cells and the potential mechanisms.Methods The expression levels of miR-520a-5p in prostate cancer cells（PC-3/DU145） and normal prostate epithelial cells（RWPE-1） were measured by RT-qPCR. Western blot was used to determine whether miR-520a-5p regulated the expression of the key glycolytic enzymes hexokinase 2（HK2） and phosphofructokinase（PFKM）. The effects of miR-520a-5p on the proliferation and docetaxel resistance of prostate cancer cells were determined using cell counting kit-8. Glucose consumption and lactic acid production in the culture supernatant were assessed by enzyme-linked immunosorbent assay. According to the transfection， all cells were divided into 4 groups： minic group，minic NC group，inhibitor group and inhibitor NC group.Results miR-520a-5p was significantly down-regulated in prostate cancer cells（P<0.001）. Compared with the mimic NC group，the mimic group showed significant decreases in the proliferative ability（P<0.001），half-maximal inhibitory concentration（IC₅₀） of docetaxel，glucose consumption level，and lactic acid production level（P<0.001） of PC-3 and DU145 prostate cancer cells. Compared with the inhibitor NC group，the inhibitor group showed significantly increased proliferative ability（P<0.001） and glucose consumption and lactic acid production levels（P<0.001） of PC-3 and DU145 prostate cancer cells. Starbase database analysis indicated that there were binding sites between miR-520a-5p and HK2 and PFKM. Western blot results showed that miR-520a-5p negatively regulated the expression of HK2 and PFKM. Inhibiting the glycolysis of prostate cancer cells by 2-DG could recover the effect of miR-520a-5p on the proliferative ability of prostate cancer.Conclusion The low expression of miR-520a-5p in prostate cancer may up-regulate the key glycolytic enzymes HK2 and PFKM to enhance aerobic glycolysis，thereby inducing resistance to docetaxel in prostate cancer cells.