Construction of Nfic gene 3′UTR dual luciferase reporter vector and targeting verification between Nfic and miR-20a / 天津医药

ABSTRACT

Objective To construct a luciferase reporter vector containing the 3′untranslated region (3′UTR) of nuclear factor I-C (nuclear factor I-C, Nfic), and apply dual luciferase reporter gene system to determine the association between microRNA-20a (miR-20a) and its potential target gene Nfic. Methods The potential complementary binding sites of miR-20a and Nfic were predicted by Targetscan. The 3′UTR of Nfic fragment amplified by PCR was cloned into luciferase reporter vector MIR- Report Luciferase. The luciferase reporters containing 3′ UTR of Nfic and miR- 20 mimics (experimental group) or NC mimics (control group) were co-transfected into 293-AD cells. Cells were collected, and then dual-luciferase reporter assay was performed to detect the luciferase activity of the two groups of cells, consequently the relationship between miR-20a and Nfic was identified. The miR-20a mimics and NC mimics were transfected into marrow stromal cell line ST2 respectively. The total cell lysates were collected, and the expression level of NFIC was detected by Western blotting assay. Results Results of double enzyme digestion and DNA sequencing showed that sequence of luciferase reporter vector was correct. miR-20a specificity bounded to Nfic 3′UTR and inhibited the luciferase activity of the reporter construct (P<0.05). Western blotting assay showed that the NFIC protein level was obviously down-regulated in ST2 cells after the transfection of miR-20a mimics compared with that of control. Conclusion The luciferase reporter vector containing the 3′UTR of Nfic is constructed successfully, which confirms that miR-20a can direct effect on Nfic3′UTR and repress its luciferase activity.