摘要
目的:观察miR-512-3p在浸润性乳腺癌和癌旁组织中的表达,以及对乳腺癌细胞系MDA-MB-231增殖、凋亡、周期和克隆形成能力的影响,并分析其可能机制。方法:运用荧光定量PCR检测浸润性乳腺癌和癌旁组织中的miR-512-3p的表达量。运用MTT法、流式细胞仪和平板克隆形成实验分析miR-512-3p对细胞增殖、凋亡、周期和克隆形成能力的影响。通过Tar getScan、PicTar和miRanda软件寻找miR-512-3p可能靶基因,应用RT-PCR和Western blot技术进行验证。结果:乳腺癌组织中miR-512-3p表达明显低于正常组织(P<0.05)。MTT试验中,72 h浓度为100 nmol/L时,miR-512-3p对乳腺癌细胞增殖的抑制作用最明显,抑制率为45.38%。miR-512-3能明显诱导细胞发生早期凋亡(9.32±0.41)%,与空白对照组(3.1±0.54)%,负对照组(2.9±0.39)%比较,差异有统计学意义(P<0.05)。与空白对照组比较,G0/G1期细胞明显增多,G2/M期细胞明显减少(P<0.05)。平板克隆试验显示过表达miR-512-3p可显著抑制细胞的克隆形成能力,并与对照组差异性显著。荧光定量PCR和Western bolt实验结果显示100 nmol/L miR-512-3p显著抑制c-FLIP mRNA和蛋白表达水平。结论:miR-512-3p在乳腺癌组织中表达明显低于正常组织,其可能通过作用于下游基因c-FLIP抑制乳腺癌细胞株MDA-MB-231的增殖和克隆形成能力,诱导凋亡,其在肿瘤发生发展中可能担任着抑癌基因的角色,可能成为未来乳腺癌诊断和治疗的一个新的靶点。
Objective：This study aims to investigate the expression pattern of miR-512-3p in breast cancer and noncancerous paired specimens as well as the effects of miR-512-3p on the proliferation, apoptosis, cell cycle, and cloning of MD-MBA-231 breast cancer cells. The study also aims to identify the miR-512-3p target gene. Methods：Reverse transcription-polymerase chain reaction （RT-PCR） was conducted to quantify the miR-512-3p expression in breast cancer and noncancerous paired specimens. Methylthiazol tetrazolium （MTT） assay, flow cytometry, and clone formation assay were used to characterize the function of miR-512-3p in breast cancer. Target prediction was performed using the TargetScan, PicTar, and miRanda software. The results were validated using RT-PCR and western blot target validation. Results：The relative expression of miR-512-3p in breast cancer specimens was significantly lower than those in normal breast specimens （P〈0.05）. MTT assay revealed that 48 h after transfection, miR-512-3p significantly repressed the proliferation of MD-MBA-231 cells at a suppression rate of 45.38%and at a concentration of 100 nmol/L. MiR-512-3p increased the percentage of early apoptotic cells in the treatment groups （9.32 ± 0.41）%compared with those in the blank controls （3.1 ± 0.54）%and in the negative controls （2.9 ± 0.39）%（P〈0.05）. Significant differences were found in the percentages of the G0/G1-and G2/M-phase cells after miR-512-3p transfection compared with those in the controls （P〈0.05）. In the cloning assay, clone formation was inhibited in the miR-512-3p-transfected groups compared with those in the control groups. RT-PCR and western blot results indicate that miR-512-3p significantly inhibited the c-FLIP mRNA and protein expression. Conclusion：MiR-512-3p expression is relatively decreased in breast cancer specimens compared with those in the normal samples. The negative effect of miR-512-3p on cell proliferation and clone formation and its positive effect on early apoptosis through c-FLIP targeting suggest that miR-512-3p acts as a tumor suppressor gene in breast cancer. Therefore, miR-512-3p may be a new target for the diagnosis and treatment of breast cancer in the future.
出处
《中国肿瘤临床》
CAS
CSCD
北大核心
2013年第19期1145-1149,共5页
Chinese Journal of Clinical Oncology
基金
国家自然科学基金项目(编号:81272240)
上海市科委基金项目(编号:STCSM10411964700)资助~~
