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《Acta Medicinae Universitatis Scientiae et Technologiae Huazhong》 2018-03
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Regulating Role of hsa-miR-4443 in Expression of TIMP2 Detected by Dual-luciferase Reporter System

Ma Tengfei;Wang Jinyan;Hou Junchen;Clinical Laboratory,The Central Hospital of Wuhan,Tongji Medical College,Huazhong University of Science and Technology;Department of General Surgery,Jiangsu Cancer Hospital & The Affiliated Cancer Hospital of Nanjing Medical University;  
Objective We aimed to determine the regulating role of hsa-miR-4443 in expression of tissue inhibitor of metalloproteinases 2(TIMP2)through constructing TIMP2-luciferase reporter gene.Methods The binding sites of hsa-miR-4443 in3′untranslated region(3′UTR)of TIMP2 were predicted by the bioinformatics analyzing tool.TIMP2 gene fragments binding hsa-miR-4443 and mutant sequences were designed and synthesized.The wild and mutant luciferase reporter gene carriers were constructed,respectively.Hsa-miR-4443 mimics or negative control of mimics were respectively co-transfected into MDA-MB-231 cells with wild or mutant luciferase reporter gene.The luciferase activity was detected to observe the influence of hsa-miR-4443 on expression of TIMP2.Results Bioinformatics analysis showed that 3 binding sites were located in 3′UTR of TIMP2.The electrophoresis and sequencing results showed that the wild and mutant luciferase reporter gene carriers of the 3 binding sites were constructed successfully.The luciferase assays revealed that hsa-miR-4443 significantly diminished luciferase activity by targeting the first binding site,whereas luciferase activity of mutant luciferase reporter gene carrier was not affected(P=0.002).Conclusion TIMP2 can be targeted by hsa-miR-4443.
【Fund】: 国家自然科学基金青年基金资助项目(No.81602551)
【CateGory Index】: R3411
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