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. 2015 Sep 29;35(5):e00251. doi: 10.1042/BSR20150153

Figure 3. miR-200a suppressed human Cx43 gene expression via directly targeting at a binding site locating at 909 in the 3′-UTR of human Cx43 gene.

Figure 3

(A) The schematic diagram of potential binding sites for functional miRs in the 3′-UTR of human Cx43 gene. (B) The 3′-UTR of human Cx43 gene containing the wt or mutated binding sequences of potential miRs was subcloned into a miR reporter vector to form the wt or mutated (mut1–mut6) reporter genes as indicated. (C). The wt or mut1 reporter plasmid (0.4 μg/ml) was co-transfected with the control miR (NC, 100 nM) or miR-23a (100 nM) into the MDA-MB-231 cells for 24 h and then the cells were harvested for luciferase assay (n=5, **P<0.01). (D) The luciferase activity of the MDA-MB-231 cells co-transfected with the reporter plasmids wt or mut (0.4 μg/ml) plus NC or miR-23b (NC, 100 nM) for 24 h (n=5, **P<0.01). (E) The luciferase activity of the MDA-MB-231 cells co-transfected with the wt or mut2 (0.4 μg/ml) plus NC or miR-200a (NC, 100 nM) for 24 h (n=5, **P<0.01). (F) The reporter plasmids wt or mut3 (0.4 μg/ml) were co-transfected with the NC or miR-200a (100 nM) into MDA-MB-231 cells for 24 h and then the cells were subjected to luciferase assay (n=5, **P<0.01). (G) The reporter plasmids wt, mut4 or mut5 (0.4 μg/ml) were co-transfected with the NC or miR-186 (100 nM) into MDA-MB-231 cells for 24 h before cell collection and luciferase assay (n=5). (H) The luciferase activity of the MDA-MB-231 cells co-transfected with the reporter gene wt or mut6 (0.4 μg/ml) plus the NC (100 nM) or miR-381 (100 nM) for 24 h (n=5).