Zhang Ting-ting, Sun Yang, Jia Cong-wei, Yu Shuang-ni, Lu Zhao-hui, Chen Jie
Department of Pathology,Peking Union Medical College Hospital, Peking Union Medical College, Chinese Academy of Medical Science, Beijing 100730, China.
Zhonghua Bing Li Xue Za Zhi. 2013 Jun;42(6):392-6. doi: 10.3760/cma.j.issn.0529-5807.2013.06.008.
To predict and verify the target gene of miR-27a in pancreatic cancer by combining the result of comparative proteome analysis.
The bioinformatics softwares of TargetScan,PicTar and miRanda were used to predict the possible target genes of miR-27a. Based on the results of comparative proteomics analysis, possible candidates of the target genes were selected. Expression vector of target gene 3'UTR was constructed, and then target gene was verified by dual-luciferase reporter assay system. The PANC-1 and BxPC-3 pancreatic cancer cells were treated with miR-27a mimics or negative control for 48 h. Western blot analysis was used to verify alterations of protein expression of the genes.
PSMA1 was selected as the candidate target gene of miR-27a by bioinformatics prediction and comparative proteome analysis. Dual-luciferase reporter assay showed that miR-27a decreased luciferase activity in cells co-transfected with pmirGLO-PSMA1-WT, compared to the negative control, although significant difference of luciferase activity was not observed in cells co-transfected with pmirGLO-PSMA1-MUT between the two groups. The protein level of PSMA1 was down-regulated in pancreatic cancer cells transfected with miR-27a mimics in comparison with pancreatic cancer cells transfected with negative control.
PSMA1 is the direct target gene of miR-27a in pancreatic cancer.
通过结合比较蛋白质组分析结果预测并验证胰腺癌中miR-27a的靶基因。
利用TargetScan、PicTar和miRanda生物信息学软件预测miR-27a可能的靶基因。基于比较蛋白质组学分析结果，选择可能的靶基因候选者。构建靶基因3'UTR表达载体，然后通过双荧光素酶报告基因检测系统验证靶基因。将PANC-1和BxPC-3胰腺癌细胞用miR-27a模拟物或阴性对照处理48小时。采用蛋白质免疫印迹分析验证基因蛋白质表达的变化。
通过生物信息学预测和比较蛋白质组分析，PSMA1被选为miR-27a的候选靶基因。双荧光素酶报告基因检测显示，与阴性对照相比，在共转染pmirGLO-PSMA1-WT的细胞中，miR-27a降低了荧光素酶活性，尽管在两组共转染pmirGLO-PSMA1-MUT的细胞中未观察到荧光素酶活性的显著差异。与转染阴性对照的胰腺癌细胞相比，转染miR-27a模拟物的胰腺癌细胞中PSMA1的蛋白水平下调。
PSMA1是胰腺癌中miR-27a的直接靶基因。
