CAJ | 학술논문

目的软件预测并筛选miR-27a可能的靶基因,结合胰腺癌比较蛋白质组学结果中表达下调的蛋白,进一步检测胰腺癌中miR-27a的靶基因并进行验证.方法采用生物信息学预测软件TargetScan、PicTar以及miranda预测miR-27a的靶基因,结合前期比较蛋白质组学的结果,筛选出miR-27a的可能靶基因；构建靶基因3'非翻译区(UTR)的表达载体,双荧光素酶报告基因检测系统验证miR-27a对靶基因的靶向作用；胰腺癌肿瘤细胞系PANC-1和BxPC-3中转染miR-27a mimics或微小RNA阴性对照序列,Western blot检测靶蛋白的表达情况.结果软件预测与蛋白质组学分析结合筛选出miR-27a的靶基因为PSMAl.双荧光素酶报告基因检测系统显示,与对照组相比miR-27a对野生型PSMA1的3'UTR具有靶向作用,实验组较阴性对照组相对活性值下降19.14％,差异具有统计学意义(P=0.016)；而对突变型PSMA1 3'UTR的靶向作用消失,实验组与阴性对照组相比差异无统计学意义(P=0.249).Western blot分析显示转染miR-27a mimics组PSMA1蛋白的相对表达量明显低于阴性对照组,PANC-1细胞实验组的PSMA1相对表达量为0.71±0.01;BxPC-3细胞实验组的PSMA1相对表达量为0.58±.0.04,P值均＜0.01.结论胰腺癌中PSMA1是miR-27a的直接靶基因.
목적 연건예측병사선miR-27a가능적파기인,결합이선암비교단백질조학결과중표체하조적단백,진일보검측이선암중miR-27a적파기인병진행험증.방법 채용생물신식학예측연건TargetScan、PicTar이급miranda예측miR-27a적파기인,결합전기비교단백질조학적결과,사선출miR-27a적가능파기인；구건파기인3'비번역구(UTR)적표체재체,쌍형광소매보고기인검측계통험증miR-27a대파기인적파향작용；이선암종류세포계PANC-1화BxPC-3중전염miR-27a mimics혹미소RNA음성대조서렬,Western blot검측파단백적표체정황.결과 연건예측여단백질조학분석결합사선출miR-27a적파기인위PSMAl.쌍형광소매보고기인검측계통현시,여대조조상비miR-27a대야생형PSMA1적3'UTR구유파향작용,실험조교음성대조조상대활성치하강19.14％,차이구유통계학의의(P=0.016)；이대돌변형PSMA1 3'UTR적파향작용소실,실험조여음성대조조상비차이무통계학의의(P=0.249).Western blot분석현시전염miR-27a mimics조PSMA1단백적상대표체량명현저우음성대조조,PANC-1세포실험조적PSMA1상대표체량위0.71±0.01;BxPC-3세포실험조적PSMA1상대표체량위0.58±.0.04,P치균＜0.01.결론 이선암중PSMA1시miR-27a적직접파기인.
Objective To predict and verify the target gene of miR-27a in pancreatic cancer by combining the result of comparative proteome analysis.Methods The bioinfonnatics 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.Results 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.Conclusion PSMA1 is the direct target gene of miR-27a in pancreatic cancer.