核因子E2相关因子2基因沉默对人前列腺癌PC-3细胞增殖、侵袭和转移的影响

 目的 探讨核因子E2相关因子2(NRF2)基因沉默对人前列腺癌PC-3细胞增殖、侵袭和转移的影响及其机制。方法 采用RT- PCR检测人前列腺上皮细胞株RWPE-1和前列腺癌细胞株PC-3中NRF2 mRNA的表达。采用脂质体转染法将NRF2 siRNA-1、NRF2 siRNA-2和NRF2 siRNA-3干扰序列转染至PC-3细胞后,Western blot和RT-PCR检测转染效果,并筛选出干扰效果最好的NRF2 siRNA序列。将体外培养的PC-3细胞分为Con组(未转染)、NC组(转染阴性对照)和NRF2 siRNA组(转染NRF2 siRNA-3),采用CCK-8法、平板克隆实验和Transwell实验分别检测各组细胞的增殖、克隆形成、侵袭和迁移能力,Western blot检测各组细胞中细胞核增殖相关抗原(Ki67)、细胞周期蛋白D1(CyclinD1)、基质金属蛋白酶9(MMP-9)、N-钙黏附素(N-cadherin)蛋白的表达。结果 与前列腺上皮细胞株RWPE-1相比,前列腺癌细胞株PC-3中NRF2 mRNA的表达水平明显升高(0.84±0.05 vs 0.22±0.03,P<0.05)。NRF2 siRNA-1、NRF2 siRNA-2和NRF2 siRNA-3干扰序列均能够明显下调PC-3细胞中NRF2蛋白(0.56±0.04,0.39±0.03,0.11±0.02 vs 0.76±0.07)和mRNA(0.80±0.05,0.52±0.03,0.26±0.03 vs 1.00±0.05)的表达,且NRF2 siRNA-3的干扰效果明显大于NRF2 siRNA-1和NRF2 siRNA-2。与Con组相比,NRF2 siRNA组细胞的增殖、克隆形成、侵袭和迁移能力存活率:24 h (82.05±5.24)% vs (99.86±5.05)%,48 h (64.57±4.85)% vs (97.28±6.36)%,72 h (45.62±3.76)% vs (94.57±5.49)%;克隆形成率:(39.35±3.26)% vs (66.52±4.85)%;侵袭细胞数:42.00±5.50 vs 85.00±7.50;迁移细胞数:58.00±3.00 vs 118.50±8.00]均明显减弱,同时Ki67、CyclinD1、MMP-9和N-cadherin蛋白的表达(Ki67:0.25±0.03 vs 0.78±0.05;CyclinD1 0.41±0.03 vs 0.85±0.06;MMP-9:0.10±0.02 vs 0.89±0.07;N-cadherin:0.22±0.02 vs 0.49±0.04)均明显降低(P<0.05);而NC组与Con组间无统计学差异(P>0.05)。结论 NRF2基因沉默可抑制人前列腺癌PC-3细胞增殖、侵袭和转移,其分子机制可能与下调Ki67、CyclinD1、MMP-9和N-cadherin蛋白的表达有关。

Effect of NRF2 gene silencing on proliferation and metastasis of human prostate cancer PC-3 cells

Objective To investigate the effect of NRF2 gene silencing on proliferation, invasion and metastasis of human prostate cancer PC-3 cells and the mechanism.Methods The expressions ofNRF2 mRNA in human prostate epithelial cell line RWPE-1 and prostate cancer cell line PC-3 were detected by RT-PCR.After the designed NRF2 siRNA-1, NRF2 siRNA-2 andNRF2 siRNA-3 interference sequences were transfected into PC-3 cells by lipofection, the bestNRF2 siRNA sequence was screened by Western blot and RT-PCR. PC-3 cells were divided into Con group (untransfected), NC group (transfected negative control) and NRF2siRNA group (transfected NRF2 siRNA-3). The proliferation, cloning, invasion and migration of each group were detected with CCK-8 method, plate cloning test and Transwell test,while the expressions of Ki67,CyclinD1, MMP-9 and N-cadherin proteins in each group were detected by Western blot.Results Compared with prostatic epithelial cell line RWPE-1, the expression of NRF2mRNA in prostate cancer cell line PC-3 was significantly increased (0.84±0.05 vs 0.22±0.03,P<0.05). The expressions of NRF2 protein(0.56±0.04,0.39±0.03,0.11±0.02 vs 0.76±0.07) and mRNA(0.80±0.05,0.52±0.03,0.26±0.03 vs 1.00±0.05) in PC-3 cells could be down-regulated by NRF2 siRNA-1, NRF2 siRNA-2 and NRF2 siRNA-3 interfering sequences,and the interference effect of NRF2 siRNA-3 was significantly greater than that of NRF2 siRNA-1 andNRF2 siRNA-2.Compared with Con group, the proliferation, cloning, invasion and migration abilitiessurvival rate:24 h (82.05±5.24)% vs (99.86±5.05)%,48 h (64.57±4.85)% vs(97.28±6.36)%,72 h (45.62±3.76)% vs(94.57±5.49)%;clone formation rate:(39.35±3.26)% vs(66.52±4.85)%;number of invasive cells:42.00±5.50 vs 85.00±7.50;number of migrating cells:58.00±3.00 vs 118.50±8.00]of NRF2 siRNA group were significantly weakened, while the expressions of Ki67, Cyclin D1, MMP-9 and N-cadherin (Ki67:0.25±0.03 vs 0.78±0.05;CyclinD1 0.41±0.03 vs 0.85±0.06;MMP-9:0.10±0.02 vs 0.89±0.07; N-cadherin:0.22±0.02 vs 0.49±0.04)were significantly decreased (P<0.05), but there was no significant difference between NC group and Con group (P>0.05).Conclusions NRF2gene silencing can inhibit the proliferation, invasion and metastasis of human prostate cancer PC-3 cells, and the molecular mechanism may be related to down-regulation of the expressions of Ki67, Cyclin D1, MMP-9 and N-cadherin proteins.