Fig. 2

MiR-125b-5p down-regulates PAD2 by directly targeting its 3’UTR and overexpression of miR-125b-5p increases the sensitivity of MCF7/TamR cells to tamoxifen. a The PAD2 3’UTR contains the binding seed sequences of miR125b-5p according to online bioinformatics analysis using Bibiserv2. Mutations of the 3′-UTR of PAD2 was used to create the mutant luciferase reporter construct. The WT and Mut-PAD2 3’UTR were separately cloned into the pGL3 luciferase reporter vector, and luciferase reporter assay showed that the activity of WT-PAD2 3’UTR, but not the mutant, was repressed by miR125b-5p overexpression. *P < 0.05. b qRT-PCR analysis showing that the endogenous miR125b-5p mRNA level was downregulated in MCF7/TamR cells compared to that in MCF7/TamS cells. ***P < 0.001. c qRT-PCR analysis confirming that miR125b-5p was stably overexpressed in MCF7/TamR cells. d PAD2 protein expression was downregulated by western blotting in miR125b-5p overexpressed MCF7/TamR cells. e Cell proliferation was not affected upon in miR125b-5p overexpressed MCF7/TamR cells by CCK8 assay, compared to the empty vector (EV Control). f Cell proliferation was inhibited in miR125b-5p overexpressed MCF7/TamR cells, in the presence of 7 μM tamoxifen, compared to the empty vector control cells. g 7 μM tamoxifen treatment on miR125b-5p overexpressed MCF7/TamR cells showing that miR125b-5p overexpression decreased colony formation. The data were presented as the mean ± SD from three independent experiments (left panel). *P < 0.05. h The mice bearing miR125b-5p overexpressed MCF7/TamR cells exhibited smaller tumors than the mice with Empty vector control cells post tamoxifen treatment (n = 3/group). The volume of tumors at indicated time after cell implantation was quantified, and the average volume was plotted. *P < 0.05