AIM: To investigate the roles of maspin in the biological behaviors of prostate cancer cells. METHODS: Specific shRNA targeting maspin gene was designed. The plasmid targeting maspin gene was constructed and lentiviral expression system was used for transfection. qRT - PCR and Western blotting were performed to identify the stable maspin - shRNA - transfected PC - 3 cells. The expression of apoptosis - related genes was analyzed by qRT - PCR. Dynamic observation of cell growth and doubling time were conducted by an xCELLigence system. The cell death upon protea-some inhibitor treatment was determined by flow cytometry analysis. The expression levels of RelA and RelB were detected by Western blotting. RESULTS: The recombinant plasmid containing maspin - shRNA was successfully constructed. Limited dilution was performed to obtain monoclonal PC — 3 — siMaspin cells. The doubling time of PC — 3 — siMaspin cells was 26. 83 h while that of PC - 3 - control cells was 37. 95 h. The mRNA expression of bcl - 2 and A20 in PC - 3 - siMaspin cells was increased, while that of bax and bim was down - regulated. The cell death rates of PC - 3 - control cells and PC - 3 - siMaspin cells after treated with MG - 132 were 27. 1% ± 5. 6% and 7. 5% ± 2. 3% at 8 h , 24. 2% ± 3. 7% and 8.2%±2.5% at24h, and 28. 7%±3. 7% and7.6%±2.5% at 36 h after treatment, respectively. RelA expression was decreased in PC - 3 - control cells treated with MG - 132 while that in PC - 3 - siMaspin cells stayed unchanged. CONCLUSION: Maspin expression is increased in androgen - independent prostate cancer PC -3 cells. Maspin silencing significantly reduces the doubling time and accelerates the cell growth. Maspin silencing markedly reduces the sensitivity of PC -3 cells to proteasome inhibitor, which may be linked to the abolishment of RelA degradation.%目的:探讨maspin影响前列腺癌细胞生物学行为的作用机制.方法:设计并合成靶向maspin基因的特异性shRNA,构建靶向maspin的shRNA慢病毒表达质粒并进行细胞转染.采用qRT-PCR和Western blotting法检测PC-3细胞转染重组质粒后maspin mRNA和蛋白质水平的变化以建立稳定转染maspin-shRNA的细胞系.采用qRT-PCR检测转染重组质粒对PC-3细胞中凋亡相关基因的影响.采用xCELLigence系统实时动态观察转染重组质粒前后细胞的生长并计算倍增时间,结合流式细胞术检测蛋白酶体抑制剂细胞毒性作用在细胞转染前后的变化.采用Western blotting法检测蛋白酶体抑制剂对核因子κB家族成员RelA和RelB表达的影响.结果:成功构建靶向maspin的shRNA慢病毒表达质粒pLL3.7-siMaspin.利用慢病毒表达系统转染并通过极限稀释法得到单克隆转染细胞PC-3-siMaspin.PC-3-siMaspin细胞的倍增时间为26.83 h,而PC-3-control细胞为37.95 h.PC-3-siMaspin细胞中bcl-2和A20 mRNA表达水平上调;而bax和bim的表达水平下调.蛋白酶体抑制剂MG-132作用PC-3-control和PC-3-siMaspin细胞8 h后细胞死亡率分别为27.1%±5.6%和7.5%±2.3%,24 h为24.2%±3.7%和8.2%±2.5%,36 h为28.7%±3.7%和7.6%±2.5%.PC-3-control细胞在MG-132作用下RelA的表达逐渐下调,而PC-3-siMaspin细胞中RelA的表达无明显变化.结论:在雄激素非依赖型前列腺癌细胞株PC-3中maspin呈高表达.Maspin表达沉默显著缩短了前列腺癌细胞的倍增时间,加快了细胞的生长与增殖.Maspin表达沉默显著下调了PC-3细胞对MG-132的敏感性,并与RelA降解的缺失相关.
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