miR-663通过靶向TGFB1对肺癌细胞A549增殖的调控

目的利用双荧光蛋白报告基因分析系统,验证miR-663的直接靶基因TGFB1,探讨miR-663促进肺癌细胞A549增殖的可能机制。方法实时定量RT-PCR检测10对肺癌组织和正常肺组织中miR-663的表达水平;利用细胞计数和集落形成实验来验证细胞转染miR-663 ASO后的A549细胞增殖。选取表达绿色荧光蛋白的质粒pcDNA3/EGFP,将TGFB1 3′UTR的一段特异性序列插入该质粒中,并与miR-663及表达红色荧光蛋白质pDsRed2-N1共同转染肺癌细胞系A549,转染后细胞提取的蛋白样品,荧光分光光度计进行定性和定量检测。结果 miR-663在肺癌组织中的表达高于在正常肺组...

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Bibliographic Details
Published in肿瘤防治研究 Vol. 39; no. 8; pp. 931 - 935
Main Author 章广玲 李宏杰 熊亚南 王梅梅 袁丽杰 朱丽华 刘志勇
Format Journal Article
LanguageChinese
Published 2012
Subjects
A549
miR-663
肺癌
转化生长因子B1
靶基因
Online AccessGet full text
ISSN1000-8578

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Summary:目的利用双荧光蛋白报告基因分析系统,验证miR-663的直接靶基因TGFB1,探讨miR-663促进肺癌细胞A549增殖的可能机制。方法实时定量RT-PCR检测10对肺癌组织和正常肺组织中miR-663的表达水平;利用细胞计数和集落形成实验来验证细胞转染miR-663 ASO后的A549细胞增殖。选取表达绿色荧光蛋白的质粒pcDNA3/EGFP,将TGFB1 3′UTR的一段特异性序列插入该质粒中,并与miR-663及表达红色荧光蛋白质pDsRed2-N1共同转染肺癌细胞系A549,转染后细胞提取的蛋白样品,荧光分光光度计进行定性和定量检测。结果 miR-663在肺癌组织中的表达高于在正常肺组织中的表达;miR-663表达明显促进了细胞A549的增殖;共转miR-663和pcDNA3/EGFP-TGFB13′UTR质粒后,绿色荧光蛋白的表达量明显低于pcDNA3和pcDNA3/EGFP-TGFB13′UTR共转组。结论 miR-663可能通过靶定靶基因TGFB1,促进了肺癌细胞A549的增殖。
Bibliography:42-1241/R
Zhang Guangling1,Li Hongjie1,Xiong Ya′nan1,Wang Meimei1,Yuan Lijie1,Zhu Lihua1,Liu Zhiyong2 1.School of Basic Medical Sciences,Hebei United University,Tangshan 063000,China;2.The Affiliated Hospital of Hebei United University
Objective To identify the miR-663 targeted gene TGFB1 using a dual fluorescent protein reporter assay system and to reveal the possible mechanism of miR-663 to promote the proliferation of A549 lung cancer cells.Methods The expression of miR-663 in lung cancer in 10 pairs of lung cancer tissues and in their adjacent normal tissues was measured using microRNA specific qRT-PCR.Than the effects of miR-663 on the proliferation of A549 cells transfected by miR-663 LNA（locked nucleic acid） was analyzed by cell growth curve and colony formation assay.A sequence of TGFB1 3′UTR（untranslated region） was inserted into the plasmid which expressed green fluorescent protein（pcDNA3/EGFP）.This plasmid（pcDNA3/EGFP-TGFB1 3′UTR） and miR-663 and the plasmid expressed red fluorescent protein（pDsRed2-
ISSN:1000-8578