摘要
目的探讨天然化合物网脊衣酸(RB)对前列腺癌LNCaP细胞的周期阻滞作用及分子机制。方法细胞经RB处理后,流式细胞术检测细胞周期的变化;溴脱氧尿嘧啶核苷(BrdU)标记法检测细胞增殖情况;Western blotting检测p53、p21CIP1(p21)及细胞周期相关蛋白的表达;实时定量PCR检测p21的mRNA水平的变化;荧光素酶报告基因检测RB对p21启动子活性的影响;转染p53突变体质粒检测p53对p21表达的影响;基因敲除p21检测其对细胞周期的作用。结果 RB可使LNCaP细胞阻滞于G0/G1期,抑制周期相关的Cyclin D、Cyclin E、Cdk 4和p-Rb表达。同时,RB可时间依赖性地诱导LNCaP细胞中野生型p53表达、促进其入核,同时上调p21的mRNA和蛋白水平。阻断p53的活性后,可抑制RB介导的p21表达及其启动子活性。而敲除p21基因,可降低RB所介导的G0/G1期阻滞,同时G2/M期细胞数量增加。结论 RB通过激活p53,在转录水平上调靶基因p21的表达,导致前列腺癌LNCaP细胞阻滞于G0/G1期,从而抑制细胞的增殖。
Abstract : Objective To investigate the effects of retigeric acid B ( RB ) on cell cycle arrest in human prostate carcino- ma LNCaP cells. Methods After exposed to RB, changes in cell cycle were monitored by flow cytometry assays. Cell proliferation was measured by incorporation of 5-Bromo-2'-deoxyuridin (BrdU) into DNA. Expressions of p53, p21 c^CIPI ( p21 ) and cell cycle related proteins in cells exposed to RB were determined by Western blotting. The mRNA level of p21 was tested by quantitative RT-PCR. Activity of p21 promoter by RB was measured by luciferase reporter assays. The effect of p53 on RB-induced p21 was assessed through interference of p53 activity with mutant p53 expression vec- tor in cells following transfection. The role of p21 in RB-mediated cell cycle arrest was determined by knockdown of p21 with small interfering RNA (siRNA). Results RB treatment led to the accumulation of LNCaP cells in the GO/ GI phase. Accordingly, the block of G0/GI phase was accompanied with decreases in Cyclin D, Cyclin E, Cdk 4 and p-Rb in cells exposed to RB in a time-dependent manner. RB significantly induced p53 expression, and promoted its nuclear location, accompanied with the increased p21 as evidenced by increases in the mRNA and protein levels. Inter- ference of p53 activity resulted in downregulation of p21 protein level and suppression of p21 promoter activity in RB-treated cells, suggesting that overexpression of p21 by RB was, at least in part, p53-dependent. Depletion of p21 by specific targeting siRNA in RB-treated cells led to the accumulation of G2/M cells, and a corresponding decrease in G0/Gl-phase fraction as compared to the scramble siRNA control, indicating the importance of RB-induced p21 in cell cycle arrest. Conclusion RB could inhibit cell proliferation via the promotion of p53/p21, overexpression of p21 in turn leading to the accumulation of LNCaP cells in the G0/G1 phase, which could contribute to RB-mediated cell prolif- eration suppression.
出处
《山东大学学报(医学版)》
CAS
北大核心
2013年第12期34-40,共7页
Journal of Shandong University:Health Sciences
基金
国家自然科学基金(81273533)
山东大学2012年大学生科技创新基金(201210422068)