CXCR4-3’-UTR双荧光素酶报告基因载体的构建及与microRNA-494的靶向作用

Construction of CXCR4-3'-UTR double luciferase reporter gene vector and its targeting effect with micro-RNA-494

目的 构建野生型与突变型CXCR4基因3’-非编码区(3’-UTR)的双荧光素酶报告基因载体,验证microRNA-494 (miR-494)对CXCR4的靶向调控作用。方法 使用生物信息学相关软件预测miR-494与CXCR4之间的靶向关系;利用PCR法扩增CXCR4的目的基因片段构建野生型与突变型CXCR4基因3’-UTR的双荧光素酶报告基因载体;将miR-494模拟物和阴性对照物分别与野生型pMIR-CXCR4-wt-3’-UTR或突变型pMIR-CXCR4-mut-3’-UTR双荧光素酶报告基因载体共同转染NRK-49F细胞,然后进一步检测相对荧光值。结果 成功构建野生型与突变型CXCR4基因3’-UTR的双荧光素酶报告基因载体,荧光结果显示加入miR-494模拟物可使野生型pMIR-CXCR4-wt-3’-UTR的荧光素酶活性降低约52%,但突变型pMIR-CXCR4-mut-3’-UTR的荧光素酶活性与对照组相比无显著变化。结论 初步证实miR-494与CXCR4之间存在靶向调控关系。

Objective To construct a dual luciferase reporter gene vector for the 3'-untranslated region(3'-UTR)of wild-type and mutant-type CXCR4 genes to verify the targeting relationship between microRNA-494(miR-494)and CXCR4.MethodsThe targeting relationship between miR-494 and CXCR4 were predicted by Bioinformatics related sofiware.The target gene fragment ofCXCR4 were amplificated by PCR to construct the CXCR4 wild-type and mutanttype dual-luciferase reporter vector.The miR-494 and negative control were co-transfected into NRK-49F cells with wild-type pMIR-CXCR4-wt-3'-UTR or mutant-type pMIR-CXCR4-mut-3'-UTR dual luciferase reporter vector,and then the relative fluorescence values were detected.Results A dual luciferase report gene vector for the 3'-UTR of wild-type and mutant-type CXCR4 genes were successfully constructed.The results of the dual luciferase reporter system show that after transfection with wild-type vector,luciferase activity decreased by about 52%in miR-494 mimics group compared with control group;but after transfection with the mutant-type vector,the luciferase activity did not change significantly.ConclusionsThese data suggests that miR-494 may have a targeted regulatory effect on CXCR4.