[1]张仁,张圣洁,张学研,等.CLPTM1L与miR494靶向关系的双荧光素酶报告实验验证[J].郑州大学学报(医学版),2015,(03):430-433.
 ZHANG Ren,ZHANG Shengjie,ZHANG Xueyan,et al.Identification of miR494 target geneCLPTM1L by luciferase reporter assay[J].JOURNAL OF ZHENGZHOU UNIVERSITY(MEDICAL SCIENCES),2015,(03):430-433.
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CLPTM1L与miR494靶向关系的双荧光素酶报告实验验证()
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《郑州大学学报(医学版)》[ISSN:1671-6825/CN:41-1340/R]

卷:
期数:
2015年03期
页码:
430-433
栏目:
应用研究
出版日期:
2015-05-20

文章信息/Info

Title:
Identification of miR494 target geneCLPTM1L by luciferase reporter assay
作者:
张仁张圣洁张学研李彤臧文巧
郑州大学基础医学院微生物与免疫学教研室 郑州 450001
Author(s):
ZHANG Ren ZHANG Shengjie ZHANG Xueyan LI Tong ZANG Wenqiao
Department of Immunology and Microbiology,College of Basic Medical Sciences, Zhengzhou University, Zhengzhou 450001
关键词:
miR494 CLPTM1L 双荧光素酶报告系统
Keywords:
miR494CLPTM1Lluciferase reporter assay
分类号:
Q786
文献标志码:
A
摘要:
目的:确定miR494与CLPTM1L的靶向关系。方法:采用生物信息学方法预测miR494与CLPTM1L基因的结合位点。采用PCR技术扩增CLPTM1L基因3UTR片段,并克隆至pmirGLO载体,构建野生型及突变型重组双荧光素酶报告质粒。将培养的293T细胞分为4组,分别共转染miR494或阴性对照和pmirGLOCLPTM1LW 3UTR或pmirGLOCLPTM1LM 3UTR,检测4组细胞中荧光素酶活性。结果:酶切和测序证实成功构建了野生型及突变型重组双荧光素酶报告质粒pmirGLOCLPTM1LW 3UTR和pmirGLOCLPTM1LM 3UTR;与共转染miR494 mimics和突变型CLPTM1L 3UTR质粒的293T细胞中荧光素酶活性(1.056 4±0.163 4)、共转染阴性对照序列和突变质粒的293T细胞中荧光素酶活性(0.961 3±0.177 9)或野生型质粒的293T细胞中荧光素酶活性(0.983 4±0.001 2)相比,共转染miR494 mimics和野生型CLPTM1L 3UTR质粒的293T细胞中荧光素酶活性(0.651 6±0.136 4)明显降低(F=4.476,P=0.040),其他3组间比较差异无统计学意义(P>0.05)。结论:CLPTM1L与miR494存在靶向关系。
Abstract:
Analysis and identification of miR494 potential target gene CLPTM1L. Methods: Bioinformatics analysis suggested CLPTM1L was a target of miR494. PCR was performed to obtain wildtype and mutant CLPTM1L 3UTR fragments, which were cloned into pmirGLO and constructed the recombinant plasmids pmirGLOCLPTM1LW and pmirGLOCLPTM1LM, respectively. 293T cells were cultured. Two recombinant plasmids were cotransfected into 293T cells with the miR494 mimics or scrambled oligonucleotide(negative control), and the relative luciferase activity was determined by luciferase reporter assay. Results: With identification of restriction enzyme digestion and sequencing, the sequences of CLPTM1LW 3UTR and CLPTM1LM 3UTR were cloned into pmirGLO successfully.Compared with the group of miR494 mimics cotransfected with pmirGLOCLPTM1LM(1.056 4±0.163 4) and the groups of miRNC cotransfected with pmirGLOCLPTM1LW(0.983 4±0.001 2) or pmirGLOCLPTM1LM(0.961 3±0.177 9), the relative luciferase activity in group of miR494 mimics cotransfected with pmirGLOCLPTM1LW showed a significant decrease(0.651 6±0.136 4;F=4.476,P=0.040). No difference was found among the aforementioned three groups(P>0.05).Conclusion: CLPTM1L is a target gene of miR494.

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备注/Memo

备注/Memo:
通信作者,女,1979年8月生,博士,副教授,研究方向:食管癌病因学,Email: zangwenqiao@sina.com
更新日期/Last Update: 1900-01-01