miR-25在人胃癌组织中的表达分析及其靶基因TOB1的鉴定

黎伯胜; 左钱飞; 肖斌; 邹全明; 郭刚

doi:10.3969/j.issn.1000-8179.2013.09.010

miR-25在人胃癌组织中的表达分析及其靶基因TOB1的鉴定

Analysis of miR-25 expression profile in human gastric cancer tissue and identification of TOB1 as its target gene

摘要

摘要:
目的分析miR-25在胃癌组织中的表达水平并鉴定其新的靶基因。
方法选取10例胃癌患者的胃癌及癌旁正常组织样本, 用qRT-PCR法检测miR-25的表达水平。利用miRNA靶基因预测数据库预测miR-25的靶基因。构建荧光素酶载体(pMIR-TOB1)及绿色荧光蛋白报告载体(pMIR-GFP-TOB1)(含有miR-25结合位点的TOB1的3'UTR片段), 利用荧光素酶活性实验和GFP荧光强度报告实验鉴定miR-25的预测靶基因(TOB1)。qRT-PCR法和Western blot法分别检测TOB1的mRNA和蛋白质的表达水平。
结果 miR-25在80%(8/10)的胃癌组织样本中表达上调。miR-25模拟物显著抑制了荧光素酶的活性(P < 0.01)和GFP荧光强度。miR-25模拟物显著下调了AGS细胞中TOB1的mRNA(P < 0.05)和蛋白质的表达。
结论 miR-25在人胃癌组织中表达上调, miR-25能结合TOB1的3'UTR并下调TOB1的mRNA和蛋白质的表达。

Abstract:
Objective This study aims to analyze the miR-25 expression profile in gastric cancer(GC) tissue and identify its novel target gene.
Methods Ten paired GC tissues and adjacent normal tissues were included in this study.MiR-25 expression levels were measured via quantitative reverse transcription polymerase chain reaction(qRT-PCR).The target genes of the miR-25 were predicted in the miRNA Target Database.Luciferase(pMIR-TOB1) and GFP reporters(pMIR-GFP-TOB1), which contain the binding site of miR-25 to TOB1 3'UTR, were constructed.The predicted target gene(TOB1) of miR-25 was identified via luciferase activation and GFP expression assays.The mRNA or protein expression of TOB1 was detected via qRT-PCR or Western blot, respectively.
Results Increased expression of miR-25 was observed in eight GC tissues(80%).miR-25 mimics significantly inhibited luciferase activation(P < 0.01) and GFP expression in the HEK293 cells, and significantly decreased the mRNA(P < 0.05) and protein expression levels of TOB1 in the AGS cells.
Conclusion miR-25 is significantly increased in human GC tissue.Moreover, MiR-25 can significantly repress the mRNA and protein expression of TOB1 by directly targeting 3'UTR.

HTML全文

参考文献(15)

施引文献

资源附件(0)