微小核糖核酸-148a调控胰腺癌细胞中ErbB3的表达

Expression of ErbB3 in pancreatic cancer cell line regulated by microRNA-148a

目的 在胰腺癌细胞中寻找并验证微小核糖核酸（miRNA）-148a的下游调控基因.方法 运用生物信息学方法预测miRNA-148a调控的目的基因,构建含目的基因的3’端非翻译区（3＇-UTR）的荧光素酶报告质粒,用脂质体转染miRNA-148a类似物和抑制剂进入胰腺癌细胞BXPC-3,检测荧光素酶活性,确定miRNA-148a是否直接与靶基因3’-UTR结合.同时改变胰腺癌细胞BXPC-3中miRNA-148a表达水平,用Western免疫印迹法在蛋白水平检测靶基因ErbB3表达的变化.统计学处理采用单因素方差分析.结果 生物信息学发现ErbB3和miRNA-148a上有一个保守的结合靶位点.miRNA-148a直接与ErbB3的3’-UTR结合,使荧光素酶活性下降到阴性对照的[-（25.00±4.70）％,F=4.66,P＜O.01]；过表达miRNA-148a后,BXPC-3细胞中ErbB3蛋白水平灰度值下降到阴性对照的[（26.16±4.69）％,F=6.563,P＜O.05].结论 miRNA-148a在胰腺癌细胞株BXPC-3中直接靶向并调控ErbB3的表达.

Objective To look for and confirm the downstream regulated gene of microRNA （miRNA）-148a in pancreatic cancer cell line.Methods The target gene regulated by miRNA-148a was predicted through bio-informatics analysis.The plasmid containing desired gene and with luciferase 3＇-untranslated region （3＇-UTR） reporter was constructed.Pancreatic cancer cells BXPC-3 were transfected with analogs and inhibitors of miRNA-148a by liposomes.The activity of luciferase was measured to determine whether miRNA-148a directly connected with desired gene.The expression level of miRNA-148a was changed in BXPC-3 cells,and the changes of target gene v-verb-b2 erythroblastic leukemia viral oncogene homolog 3 （awian） （ErbB3） expression were detected by Western blot at protein level.The data were analyzed by one way ANOVA.Results There was a conservative binding site of ErbB3 with miRNA-148a detected by bio-informatics analysis,miRNA-148a directly combined with ErbB3 and the activity of luciferase decreased to （25.00＋47.00） % of the negative control （F=4.66,P〈 0.01）.After miRNA-148a overexpression,the gray value of ErbB3 expression in BXPC-3 cells decreased to （26.16±4.69）% of control group （F=6.563,P〈0.05）.Conclusion miRNA-148a directly targeted and regulated the expression of ErbB3 in pancreatic cancer cell line BXPC-3.