| 人miRNA let 7a2质粒的构建及其表达 |
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| 引用本文： | 关恒云,张菊,张鹏举,陈蔚文,刘闻闻,于春晓,胡晓燕,姜安丽
.人miRNA let 7a2质粒的构建及其表达[J].山东大学学报(医学版),2009,47(2):1-5. |
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| 作者姓名： | 关恒云 张菊 张鹏举 陈蔚文 刘闻闻 于春晓 胡晓燕 姜安丽
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| 作者单位： | 关恒云,张菊,张鹏举,陈蔚文,胡晓燕,姜安丽,GUAN Heng-yun,ZHANG Ju,ZHANG Peng-ju,CHEN Wei-wen,HU Xiao-yan,JIANG An-li(山东大学,医学院生物化学与分子生物学研究所,济南,250012);刘闻闻,LIE Wen-wen(山东大学,附属省立医院耳鼻喉头颈外科,济南,250021);于春晓,YU Chun-xiao(山东大学,附属省立医院内分泌科,济南,250021)
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| 摘 要： | 目的构建人miRNA let 7a2真核表达质粒，检测其在肺癌细胞A549中的表达。方法以人肺腺癌A549细胞总RNA为模板，将RT PCR扩增let 7a2的前体（pre let7a2）序列, 克隆至pSilencer4.1 CMV neo表达载体中，构建pSilencer4.1 let7a2重组质粒，转染A549细胞,采用RT PCR法检测pre let7a2的表达；构建let 7a2靶序列-报道基因融合质粒pMIR Report let7a2T，与pSilencer4.1 let7a2质粒共转染A549细胞，测定相对荧光素酶活性,以检测成熟let7a2在A549细胞中的表达及作用；采用MTT比色法检测pSilencer4.1 let7a2转染对A549细胞增殖的影响。结果构建的人let 7a2真核表达质粒pSilencer4.1 let7a2和let7a2靶序列-报道基因融合质粒pMIR Report let7a2T经酶切及测序鉴定正确；pSilencer4.1 let7a2质粒转染A549细胞后，RT PCR检测pre let7a2表达较对照组明显增强；MTT比色法显示let 7a2对A549细胞增殖有抑制作用。pSilencer4.1 let7a2 质粒和pMIR Report let7a2T质粒共转染A549细胞后，通过报告基因检测相对荧光素酶活性较对照组降低，显示let 7a2表达质粒转染A549细胞后，可有效表达let 7a2并转变为成熟的具有生物学活性的let 7a2。 结论成功构建了人let 7a2真核表达质粒,并在肺腺癌细胞A549中有效表达。
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| 关 键 词： | 肺肿瘤 基因表达调控 miRNA let 7a2 |
Construction and expression of eukaryotic expression plasmid of microRNA let-7a2 in lung cancer cells |
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| GUAN Heng-yun,ZHANG Ju,ZHANG Peng-ju,CHEN Wei-wen,LIU Wenwen,YU Chun-xiao,HU Xiao-yan,JIANG An-li.Construction and expression of eukaryotic expression plasmid of microRNA let-7a2 in lung cancer cells[J].Journal of Shandong University:Health Sciences,2009,47(2):1-5. |
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| Authors: | GUAN Heng-yun ZHANG Ju ZHANG Peng-ju CHEN Wei-wen LIU Wenwen YU Chun-xiao HU Xiao-yan JIANG An-li |
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| Institution: | 1.Institute of Biochemistry and Molecular Biology;School of Medicine;Shandong University;Jinan 250012;China;2.Department of Otolaryngology-Head and Neck Surgery;Provincial Hospital Affiliated to Shandong University;Jinan 250021;3.Department of Endocrinology;China |
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| Abstract: | Objective To construct a recombinant eukaryotic expression plasmid of microRNA let-7a2 and express it in lung cancer A549 cells.Methods let-7a2 precursor sequence was amplified by RT-PCR using RNA from A549 cells and was cloned into the pSilencer4.1-CMV neo-expression vector to produce the recombinant pSilencer4.1let7a2.Then the recombinant pSilencer4.1-let7a2 was transfected into lung cancer A549 cells,and the pre-let7a2 expression was verified by RT-PCR.According to the miRBase Targets database,the target... |
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| Keywords: | miRNA let-7a2 |
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