Zhou P, Ma L, Zhou J, Xu J J, Liu F, Guo F
Central Laboratory, the First Affiliated Hospital of Soochow University, Suzhou 215006, China.
Jiangsu Institute of Hematology, Suzhou 215006, China.
Zhonghua Zhong Liu Za Zhi. 2017 May 23;39(5):325-331. doi: 10.3760/cma.j.issn.0253-3766.2017.05.002.
To explore the effect and mechanism of over-expression of miR-17-92 gene cluster on the biological characteristics of prostate cancer cells. DU145 cells were transfected with miR-17-92 gene expression plasmid and clones with stable ectopic miR-17-92 overexpression were established. The cell viabilities of DU145-17-92 and DU145-control cells were monitored by xCELLigence system. Cell proliferation and apoptosis were analyzed by Ki-67 and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Cell cycle was detected by flow cytometry. Expression levels of proteins involved in apoptosis and Akt pathway were determined by western blotting. xCELLigence RTCA array data showed that the growth rate of DU145-17-92 cells was significantly higher than that of DU145-control cells after 24 h of seeding (<0.01). The Ki-67-positive rates of the DU145-control group at 24, 48 and 72 hours were (56.57±1.68)%, (85.48±0.26)% and (90.85±2.08)%, respectively. While the Ki-67 positive rates of the DU145-17-92 group at the desired time points were (73.64±0.68)%, (93.43±1.23)% and (97.36±0.86)%, respectively, with a statistically significant difference at 24 hours (<0.01). The percentages of apoptotic cells of the DU145-control group at 24, 48 and 72 hours were (6.76±0.09)%, (14.51±0.86)% and (20.73±1.64)%, respectively, while the apoptotic percentages of the DU145-17-92 group were (1.86±0.15)%, (7.90±0.40)% and (4.92±0.48)%, respectively. The percentages of apoptotic cells of the DU145-control group at different time were significantly higher than those of DU145-17-92 group (<0.01 for all). The result of western blotting showed that the protein expression levels of Bcl-2 interacting mediator of cell death (BIM) and phosphatase and tensin homolog deleted on chromosome ten (PTEN) in DU145-control cells were 0.83±0.00 and 0.91±0.00, respectively, significantly higher than 0.16±0.00 and 0.13±0.00 of DU145-17-92 cells (both <0.01). Overexpression of miR17-92 induced the phosphorylation of protein kinase B (Akt) at Ser473 while no appreciable effect on the phosphorylation of Akt at Thr308. The phosphorylated level of extracellular regulated protein kinases (ERK) in DU145-control cells was 0.21±0.01, significantly lower than 0.72±0.01 of DU145-17-92 cells (<0.01). Overexpression of miR-17-92 gene plays a pivotal role in growth, proliferation, apoptosis and cell cycle of DU145 cells through down-regulation of apoptotic protein BIM and tumor suppressor PTEN and activation of Akt and ERK signaling pathway.
探讨miR-17-92基因簇过表达对前列腺癌细胞生物学特性的影响及机制。用miR-17-92基因表达质粒转染DU145细胞，建立稳定异位过表达miR-17-92的克隆。通过xCELLigence系统监测DU145-17-92和DU145对照细胞的细胞活力。采用Ki-67和末端脱氧核苷酸转移酶dUTP缺口末端标记（TUNEL）法分析细胞增殖和凋亡情况。通过流式细胞术检测细胞周期。采用蛋白质印迹法检测凋亡相关蛋白和Akt信号通路中相关蛋白的表达水平。xCELLigence RTCA阵列数据显示，接种24小时后，DU145-17-92细胞的生长速率显著高于DU145对照细胞（<0.01）。DU145对照组在24、48和72小时的Ki-67阳性率分别为（56.57±1.68）%、（85.48±0.26）%和（90.85±2.08）%。而DU145-17-92组在相应时间点的Ki-67阳性率分别为（73.64±0.68）%、（93.43±1.23）%和（97.36±0.86）%，24小时时差异有统计学意义（<0.01）。DU145对照组在24、48和72小时的凋亡细胞百分比分别为（6.76±0.09）%、（14.51±0.86）%和（20.73±1.64）%，而DU145-17-92组的凋亡百分比分别为（1.86±0.15）%、（7.90±0.40）%和（4.92±0.48）%。DU145对照组不同时间的凋亡细胞百分比均显著高于DU145-17-92组（均<0.01）。蛋白质印迹结果显示，DU145对照细胞中细胞死亡的Bcl-2相互作用介质（BIM）和10号染色体缺失的磷酸酶和张力蛋白同源物（PTEN）的蛋白表达水平分别为0.83±0.00和0.91±0.00，显著高于DU145-17-92细胞的0.16±0.00和0.13±0.00（均<0.01）。miR17-92过表达诱导蛋白激酶B（Akt）在Ser473位点磷酸化，而对Akt在Thr308位点的磷酸化无明显影响。DU145对照细胞中细胞外调节蛋白激酶（ERK）的磷酸化水平为0.21±0.01，显著低于DU145-17-92细胞的0.72±0.01（<0.01）。miR-17-92基因过表达通过下调凋亡蛋白BIM和肿瘤抑制因子PTEN以及激活Akt和ERK信号通路，在DU145细胞的生长、增殖、凋亡和细胞周期中起关键作用。
