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. 2018 Aug 16;8:12284. doi: 10.1038/s41598-018-30682-5

Figure 2.

Figure 2

GNMT is a direct target of miR-224. (A) GNMT had a putative response element of hsa-miR-224 as predicted by miRWalk and miRTar algorithms. The sequence of miR-224 was aligned with the CDS of GNMT in human (Homo sapiens), chimpanzee (Pan troglodytes), monkey (Macaca mulatta), mouse (Mus musculus), rat (Rattus norvegicus), cow (Bos taurus) and dog (Canis familiaris). (B) Schematic representation of dual luciferase reporter plasmids containing wild type (psiCHECK2-GNMT-WT, psi-WT) and mutant GNMT (psiCHECK2-GNMT-MT, psi-MT). The asterisk indicates the position of the putative miR-224 binding site, which was mutated in psi-MT reporter plasmid. (C) HEK293T cells were transfected with psi-WT or psi-MT plasmid and co-transfected with 224-mimic or NC, respectively. The miR-224 expression level of each sample was normalized with the level of RNU48. The Renilla luciferase activity was determined and normalized with firefly luciferase activity. Quantification of the data was shown as mean ± SD (n = 3). (D) The mRNA levels of GNMT in HEK293T cells co-transfected with the indicated plasmids and 50 nM 224-mimic were measured by real-time PCR and normalized with control group (upper panel). **P < 0.01; ***P < 0.001. Whole cell extracts of the transfected cells were harvested for western blot analysis (bottom panel). The normalized GNMT-Flag and GNMT level are presented below each lane.