靶向TRPV6的siRNA对前列腺癌LNCaP细胞抑制作用的体外研究

Inhibitory effect of TRPV6 silencing on prostate cancer cell line LNCaP in vitro

目的:运用RNA干扰(RNAi)技术阻断前列腺癌LNCaP细胞中瞬时感受器电位离子通道蛋白V亚家族6(TRPV6)基因的表达,探讨TRPV6基因沉默对LNCaP细胞增殖、细胞周期及凋亡的影响。方法:针对TR-PV6基因,构建2条小干扰RNA(siRNA)序列;在脂质体介导下转染前列腺癌LNCaP细胞,用RT-PCR测定TRPV6mRNA的表达;MTT法和流式细胞技术测定siRNA对LNCaP细胞增殖、细胞周期及凋亡的影响。结果:两条siRNA序列均能有效地阻断LNCaP细胞中TRPV6基因在mRNA水平上的表达(P<0.01),且mRNA表达随着转染时间的延长而减少,转染72h后TRPV6mRNA的表达减少至对照组的27%和23%;siRNA转染能显著抑制LNCaP细胞增殖,转染48h细胞增殖抑制率最高,分别为34.53%和29.32%,与转染24h和72h比较有统计学意义(P<0.05);转染48h后,siRNA转染组G0～G1期细胞增多,S期细胞数量明显减少,与空白对照组及阴性对照组相比有统计学差异(P<0.01);siRNA转染组细胞的凋亡率分别为14.45%和12.73%,显著高于空白对照组及阴性对照组3.78%和5.22%(P<0.05)。结论:针对TRPV6的siRNA在体外能有效地抑制LNCaP细胞TRPV6mRNA的转录,同时能够抑制前列腺癌LNCaP细胞增殖,使细胞周期出现G0/G1期阻滞,细胞凋亡率显著增加。

Objective：To explore the effects of the TRPV6 gene silencing by small interfering RNA （siRNA） on the proliferation,cell cycle and apoptosis of human prostate cancer LNCaP cells.Methods：We constructed two siRNA sequences （siTRPV6-1 and siTRPV6-2） targeting the TRPV6 gene and then transfected them into LNCaP cells mediated by liposome.The transcription of TRPV6 mRNA was detected by RT-PCR,and the effects of siRNA on the proliferation,cell cycle and apoptosis of the LNCaP cells were determined by MTT and flow cytometry.Results：Both siTRPV6-1 and siTRPV6-2 significantly suppressed the expression of TRPV6 mRNA in the LNCaP cells,and the expression was decreased with the extension of time,by 73 and 77% respectively at 72 h after transcription with siTRPV6-1 and siTRPV6-2 as compared with the blank control group （P 0.01）.The proliferation inhibition rates were the highest （34.53 and 29.32%） at 48 h in comparison with 24 and 72 h （P0.05）.The number of cells was significantly increased in the G0 and G1 phases and decreased in the S phase after siTRPV transfection （P 0.01）.The apoptosis rates of LNCaP cells were 14.45 and 12.73% respectively at 48 h after transfected with siTRPV6-1 and siTRPV6-2,significant higher than in the blank control and negative control groups （P 0.05）.Conclusion：TRPV6-targeted siRNA can effectively inhibit the transcription of TRPV6 mRNA,inhibit the proliferation of LNCaP cells,arrest their cycles in the G0 and G1 phases,and induce their apoptosis.