摘要
目的 观察微小RNA-101（miR-101）通过靶向抑制果蝇zeste基因增强子同源物2（EZH2）基因对人前列腺癌PC-3细胞生物学行为的影响.方法 应用miR-101模拟物转染PC-3细胞;实时定量聚合酶链反应（Real-time PCR）和Western blot法检测转染后miR-101和EZH2的表达;噻唑蓝（MTT）比色法检测细胞增殖、流式细胞仪检测细胞周期;膜联蛋白V（Annexin V）/碘化丙锭（PI）双染色法测量细胞凋亡率;划痕实验检测细胞迁移能力;Transwell侵袭实验检测细胞侵袭力.结果 miR-101在PC-3细胞中的表达明显低于RWPE-1细胞,EZH2 mRNA和蛋白在PC-3细胞中的表达显著高于RWPE-1细胞,miR-101的表达量与EZH2表达量呈负相关（P＜0.05）.miR-101模拟物转染PC-3细胞后,细胞中的miR-101表达上调（P＜0.05）,而EZH2 mRNA和蛋白表达水平也明显降低（P＜0.05）.MTT法显示：转染后48、72、96 h miR-101模拟物转染组细胞增殖明显低于两个对照组（P＜0.05）;流式细胞仪测定结果显示：转染后48 h,miR-101组发生了G0/G1期阻滞（P＜0.05）;细胞凋亡率检测结果显示：空白对照组、阴性对照组及miR-101模拟物转染组PC-3细胞凋亡率分别为（2.53±0.36）％、（2.71±0.42）％、（9.84±0.83）％,转染组凋亡率明显高于两个对照组（P＜0.01）.划痕实验显示：miR-101模拟物转染组细胞迁移能力明显低于两个对照组（P＜0.01）.Transwell侵袭实验结果显示：miR-101组穿过Matrigel凝胶的细胞数显著少于两个对照组（P＜0.01）.结论 miR-101负向调控EZH2基因,上调miR-101可抑制PC-3细胞中EZH2的表达,具有显著的肿瘤抑制效应.
Objective To investigate the regulatory effect of microRNA （miR）-101 on the biological features of human prostate cancer cell line PC-3 by targeting enhancer of zeste homolog 2 （EZH2）.Methods The expression of miR-101 and EZH2 in prostate cancer cell lines （PC-3,and LNCaP） and normal prostate epithelial RWPE-1 cell line was detected by real-time quantitative polymerase chain reaction （Real-time PCR） and Western blotting respectively.Dual luciferase reporter gene assay was employed to determine if miR-101 could target EZH2 through binding to 3＆#39; untranslated region （3 ＆#39;-UTR） of EZH2 mRNA.PC-3 cells were transfected with synthetic miR-101 mimic.Methyl thiazol tetrazolium （MTT） assays were used to measure the proliferation of the PC-3 cells.Flow cytometry was used to analyze the cell cycle distribution.Annexin V/propidium iodide （PI） double staining was used to measure cell apoptosis.The wound scrape assays were used to measure the cell motility.Transwell invasion assays were used to analyze the invasiveness in vitro.Results The expression levels of miR-101 in PC-3 and LNCaP cell lines were lower than in the RWPE-1 cell line,while the expression levels of EZH2 mRNA and protein were higher.There was a negative correlation between the expression of miR-101 and EZH2 （P 〈 0.05）.Dual luciferase reporter gene assay confirmed EZH2 was one of regulatory genes by miR-101.After miR-101 mimic was transfected into prostate cancer PC-3 cells,the expression of miR-101 was up-regulated （P 〈 0.05）,and that of EZH2 mRNA and protein was down-regulated significantly （P 〈 0.05）.The growth rate in miR-101 group was lower significantly than that in two control groups at 48,72 and 96 h after transfeetion （P 〈 0.05）.Flow eytometry analysis showed there was G0/G1 phase arrest in miR-101 group compared to two control groups （P 〈 0.05）.Cell apoptosis rate in transfected experimental group with miR-101 mimics （miR-101 group）,blank control group and negative control group at 48 h after transfection was （9.84 ± 0.83） %,（2.53 ± 0.36） % and （2.71 ± 0.42） % respectively.The apoptosis rate in miR-101 group was higher than two control groups （P 〈 0.01）.Scarification test demonstrated that the migration of cells in miR-101 group was more slowly than two control groups （P 〈 0.01）.Transwell test showed that the number of PC-3 cells penetrating the Matrigel gel in miR-101 group was significantly less than two control groups （P 〈 0.01）.Conclusion EZH2 may be one of target genes of miR-101 that plays a negative regulatory role.The upregulation of miR-101 can lead to the lower expression of EZH2 in PC3 cells,suggesting miR-101 exerts significantly inhibitory effect on prostate cancer.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2014年第10期2255-2258,共4页
Chinese Journal of Experimental Surgery
基金
国家自然科学基金资助项目（81172706）
