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

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.