不同双荧光素酶方法对检测胃癌相关miRNAs靶向基因TIAM1的影响  

Detection of gastric cancer related miRNAs targeting TIAM1 gene based on different double luciferase assay methods

作　　者：史艳芬[1] 胡高峰 罗杰[1] 钟定荣[1] 续薇 SHI Yan-fen;HU Gao-feng;LUO Jie(Department of Pathology,China-Japan Friendship Hospital,Beijing 100029,China)

机构地区：[1]中日友好医院病理科,北京100029 [2]北京医院国家老年医学中心国家卫生健康委临床检验中心中国医学科学院老年医学研究院,北京100730 [3]吉林大学第一医院检验科,长春130021

出　　处：《中日友好医院学报》2021年第1期28-31,共4页Journal of China-Japan Friendship Hospital

基　　金：国家自然科学基金(81672106)。

摘　　要：目的:探讨不同双荧光素酶实验方法对检测胃癌相关miRNAs靶向基因TIAM1的影响。方法:从胃癌细胞系BGC823基因组中提取T-细胞淋巴瘤侵袭转移诱导基因(TIAM1)的3’非编码区(3’-UTR)片段全长,将TIAM1-3’UTR构建在2种不同的荧光素酶基因质粒上,选择8个miRNAs进行双荧光素酶实验研究。结果:miR-10b的抑制物使2种质粒的相对荧光素酶活性均升高;miR-651和miR-653的抑制物使2种质粒的相对荧光素酶活性变化完全相反;miR-10b-5p的抑制物和类似物(mimic)分别使psi-TIAM1-3’UTR的相对荧光素酶活性增高和减弱;而miR-372-3p、miR-373-3p和miR-653-5p的抑制物和类似物处理组荧光素酶活性均升高,miR-335-3p的抑制物和类似物处理组荧光素酶活性均减低。结论:进行双荧光素酶实验时psi-TIAM1-3’UTR这类2种基因位于同一质粒且不以Renilla作为内参的质粒较为合适,并发现miR-10b-5p能够直接靶向作用于TIAM1-3’UTR。Objective:To investigate the effects of different dual-luciferase assay methods on the detection of gastric cancer related miRNAs targeting T-cell lymphoma invasion and metastasis gene(TIAM1).Methods:The full-length 3’UTR fragment of TIAM1 was extracted from the genome of a gastric cancer cell line BGC823.TIAM1-3’ UTR was constructed on two different luciferase plasmids.We selected eight miRNAs for the dualluciferase experiment.Results:After treatment with miR-10b-inhibitor,luciferase activity of both plasmids was increased.The changes of luciferase activity of the two plasmids after treatment with miR-651-inhibitor or miR-653-inhibitor were completely opposite.The luciferase activity was increased and decreased after the treatment with miR-10b-5p inhibitor and mimic,respectively.However,the luciferase activity of miR-372-3 p,miR-373-3p and miR-653-5p was increased both in the inhibitor and mimic treatment groups,while luciferase activity of miR-335-3 p was decreased both in the inhibitor and mimic treatment groups.Conclusion:It is appropriate to use plasmids like psi-TIAM1-3’UTR which contains two kinds of luciference genes and the Renilla gene was not used as internal reference for the dual-luciferase assay.MiR-10b-5p was found to be able to directly target TIAM1-3’UTR.

关 键 词：双荧光素酶实验 T-细胞淋巴瘤侵袭转移诱导基因 微小RNA 

分 类 号：R735.2[医药卫生—肿瘤]