摘要目的 慢病毒介导短发卡RNA(shRNA)靶向抑制前列腺癌细胞株LnCaP、VCaP 细胞的抑制神经前体细胞表达下调因子8(NEDD8)的表达,观察其对雄激素受体(AR)、前列腺特异性抗原(PSA)的表达以及细胞生物学行为的影响.方法 构建NEDD8短发夹RNA(NEDD8-shRNA)稳定转染的LnCaP、VCaP 细胞株.实时荧光定量聚合酶链反应(FQ-PCR)及Western blot法检测NEDD8、AR及PSA mRNA及蛋白表达.细胞计数试剂盒(CCK-8)法检测细胞增殖、流式细胞术检测细胞凋亡、TranswellTM 实验检测细胞侵袭能力.结果 NEDD8-shRNA慢病毒转染后,LnCaP、VCaP 细胞NEDD8 mRNA表达量分别是空白对照组的(0.11±0.03)、(0.16±0.04)倍,AR mRNA表达量分别为(0.52±0.04)、(0.42±0.05)倍,PSA mRNA的表达量分别为(0.49±0.04)、(0.59±0.06)倍.慢病毒转染后,LnCaP、VCaP 细胞NEDD8蛋白相对表达量分别为(11.26±2.65)%、(6.26±2.04)%,AR蛋白相对表达量分别为(30.36±4.35)%、(15.36±3.37)%,PSA蛋白相对表达量分别为(13.37±2.70)%、(11.36±2.67)%.与对照组比较,NEDD8、AR、PSA mRNA及蛋白表达均显著下降(P=0.000).慢病毒转染后,与对照组比较,LnCaP、VCaP 细胞增殖显著抑制;转染后 LnCaP、VCaP细胞株凋亡率分别为(15.08±1.23)%、(20.37±1.76)%,与对照组比较显著增加(P=0.001、0.000);穿膜细胞数显著减少(P=0.000、0.004),分别为(55.5±12.5)、(60.8±11.3) 个.结论 NEDD8可能在转录水平参与了对AR表达调控,通过抑制NEDD8基因能有效抑制LnCaP、VCaP中AR表达及细胞的增殖、侵袭能力,诱导细胞凋亡.

abstractsObjective To explore the effect of lentivirus-mediated short hairpin RNA (shRNA) silencing neural precursor cell-expressed developmentally down regulated 8 (NEDD8) gene on androgen receptor (AR),prostate specific antigen (PSA),proliferation,apoptosis and invasion of human prostate cancer LNCap and VCaP cells.Methods LNCap and VCaP cells were transfected with lentivirus-mediated NEDD8-shRNA.The mRNA and protein expression of NEDD8,AR and PSA was detected by real-time fluorescent quantitative polymerase chain reaction (FQ-PCR) and Western blotting,respectively.The proliferation,apoptosis and invasion abilities were evaluated by cell counting kit-8 (CCK-8) assay,flow cytometry and Transwell TM assay,respectively.Results After transfection with NEDD8-shRNA,NEDD8 mRNA expression in LnCaP and VCaP was (0.11±0.03) fold and (0.16±0.04) fold,respectively,as compared to the blank control group;AR mRNA expression was (0.52±0.04) fold and (0.42±0.05) fold;PSA mRNA expression was (0.49±0.04) fold and (0.59±0.06) fold.NEDD8,AR and PSA protein expression in LnCaP and VCaP was (11.26±2.65)% and (6.26±2.04)%,(30.36±4.35)% and (15.36±3.37)%,and (13.37±2.70)% and (11.36±2.67)%,respectively.As compared with the control groups,the mRNA and protein expression levels of NEDD8,AR and PSA were significantly inhibited after transfection with NEDD8-shRNA (P=0.000).As compared with the control groups,the proliferation of LnCaP and VCaP was significantly inhibited after transfection with NEDD8-shRNA.The apoptosis rate of LNCap and VCaP cells was (15.08±1.23)% and (20.37±1.76)% respectively,which was significantly enhanced as compared with the control groups (P=0.001,P=0.000).After transfection,the number of LnCaP and VCaP cells invading the Matrigel was (55.5±12.5) and (60.8±11.3) respectively,which was significantly reduced as compared with the control groups (P=0.000,P=0.004).Conclusion NEDD8 may downregulate AR at the transcriptional level.The lentivirus-mediated shRNA silencing NEDD8 gene could inhibit the proliferation and invasion,and promote apoptosis of LNCap and VCaP cells.