过表达miR-17—92基因簇对前列腺癌细胞生物学特性的影响及机制  

Effects of overexpression of miR-17-92 gene cluster on the biological characteristics of prostate cancer cells and its mechanism

作　　者：周鹏[1] 马亮 周珺[1] 徐晶晶[1] 刘菲[1] 国风[1] 

机构地区：[1]苏州大学附属第一医院临床检测中心,215006 [2]江苏省血液研究所,苏州215006

出　　处：《中华肿瘤杂志》2017年第5期325-331,共7页Chinese Journal of Oncology

基　　金：国家自然科学基金（81172433、81400154）;江苏省自然科学基金（BK20151211）

摘　　要：目的探讨过表达miR-17-92基因簇对前列腺癌细胞生物学特性的影响及潜在机制。方法采用过表达miR-17-92基因的质粒转染包装细胞，并构建过表达miR-17-92基因簇的前列腺癌DU145-17-92细胞。采用xCelligence系统实时动态监测DU145-control和DU145-17-92细胞的生长能力，采用Ki-67抗原检测技术和原位末端转移酶标记技术（TUNEL）分别检测DU145-control和DU145-17-92细胞中细胞增殖和凋亡情况，采用流式细胞术分析DU145-control和DU145-17-92细胞的细胞周期，采用Western blot检测DU145-control和DU145-17-92细胞中凋亡相关蛋白和增殖相关蛋白的表达。结果xCelligence系统监测显示，从接种24 h后，DU145-17-92细胞的生长速度显著高于DU145-control细胞（P〈0.01）。细胞培养24、48、72 h后，DU145-control组的Ki-67阳性细胞率分别为（56.57±1.68）%、（85.48±0.26）%和（90.85±2.08）%，DU145-17-92组的Ki-67阳性细胞率分别为（73.64±0.68）%、（93.43±1.23）%和（97.36±0.86）%，两组仅在培养24 h时差异有统计学意义（P〈0.01）。细胞培养24、48、72 h后，DU145-control组的细胞凋亡率分别为（6.76±0.09）%、（14.51±0.86）%和（20.73±1.64）%，DU145-17-92组的细胞凋亡率分别为（1.86±0.15）%、（7.90±0.40）%和（4.92±0.48）%，差异均有统计学意义（均P〈0.01）。Western blot检测显示，DU145-control和DU145-17-92细胞中，Bcl-2家族促凋亡蛋白（BIM）的表达水平分别为0.83±0.00和0.16±0.00, 人第10号染色体缺失的磷酸酶及张力蛋白同源物（PTEN）蛋白的表达水平分别为0.91±0.00和0.13±0.00，差异均有统计学意义（均P〈0.01）。过表达miR-17-92基因簇促进了蛋白激酶B（Akt）中丝氨酸473位点（p-Akt-473）的磷酸化，但对308位点（p-Akt-308）的磷酸化无明显影响。DU145-control和DU145-17-92细胞中磷酸化细胞外信号调节激酶（p-ERK）蛋白的表达水平分别为0.21±0.01和0.72±0.01，�ObjectiveTo explore the effect and mechanism of over-expression of miR-17-92 gene cluster on the biological characteristics of prostate cancer cells.MethodsDU145 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.ResultsxCELLigence 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 （P〈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 （P〈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 （P〈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 DU14

关 键 词：前列腺肿瘤 miR-17-92基因簇 Bcl-2家族促凋亡蛋白 人第10号染色体缺失 的磷酸酶及张力蛋白同源物 蛋白激酶B 细胞外调节蛋白激酶 

分 类 号：R737.25[医药卫生—肿瘤]