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目的:研究沉默丙酮酸激酶M2型(PKM2)对藤黄酸(GA)诱导人前列腺癌PC3细胞凋亡的影响,并探讨其潜在的作用机制。方法:设计并合成针对PKM2的3条特异性siRNA及阴性对照siRNA(si-NC)。利用脂质体将PKM2 siRNA和si-NC转染至人前列腺癌PC3细胞并检测PKM2 siRNA的沉默效率。MTT和吖啶橙/溴化乙啶(AO/EB)双重染色法分别检测沉默PKM2后GA对PC3细胞生长活性和凋亡的影响。采用实时荧光定量PCR和Western印迹法分别检测c-myc和cyclin D1的mRNA和蛋白表达水平。结果:与si-NC组比较,3条PKM2 siRNA都能够有效下调PKM2的mRNA和蛋白表达水平,其中PKM2 siRNA-1的沉默效率最高。转染PKM2siRNA-1 24 h后,PC3细胞中PKM2 mRNA和蛋白表达水平分别下调70%和85%(P<0.05)。转染PKM2 siRNA-1后给予0.5μmol/L的GA处理24 h,PC3细胞生长活性明显受到抑制(对照组的68%)且凋亡诱导增加,而且c-myc和cyclin D1基因的mRNA和蛋白的表达水平显著下调(c-myc分别为对照组的50%和35%;cyclin D1分别为对照组的60%和20%,P<0.05)。结论:抑制PKM2能够提高GA诱导人前列腺癌PC3细胞凋亡的敏感性,PKM2可能成为GA治疗前列腺癌的有效增敏靶点。
Objective: To investigate the effect of PKM2 on the apoptosis of human prostate cancer PC3 cells induced by gambogic acid (GA), and to explore its potential mechanism. Methods: Three specific siRNA against PKM2 and a negative control siRNA (si-NC) were designed and synthesized. PKM2 siRNA and si-NC were transfected into human prostate cancer PC3 cells using liposomes and the silencing efficiency of PKM2 siRNA was examined. MTT assay and acridine orange / ethidium bromide (AO / EB) double staining method were used to detect the effect of GA after silencing PKM2 on the growth activity and apoptosis of PC3 cells. The mRNA and protein expression of c-myc and cyclin D1 were detected by real-time fluorescence quantitative PCR and Western blot respectively. Results: Compared with si-NC group, all three PKM2 siRNAs could effectively down-regulate PKM2 mRNA and protein expression, of which PKM2 siRNA-1 had the highest silencing efficiency. After transfected with PKM2 siRNA-1 for 24 h, the expression of PKM2 mRNA and protein in PC3 cells were reduced by 70% and 85%, respectively (P <0.05). After treated with 0.5μmol / L GA for 24 h, the growth activity of PC3 cells was significantly inhibited (PKC 68%) and the apoptosis induction was increased. The mRNA and protein of c-myc and cyclin D1 (C-myc were 50% and 35% of the control group respectively; cyclin D1 was 60% and 20% respectively of the control group, P <0.05). CONCLUSION: Inhibition of PKM2 can increase the sensitivity of GA to induce apoptosis of human prostate cancer PC3 cells. PKM2 may be an effective target of GA in the treatment of prostate cancer.