Figure 7

Identification of candidate targets of miR-183/-96/-182 cluster miRNAs. Predicted miR-183/-96/-182 targets are listed in Additional file 4: Table S2 where their NCBI reference sequence, putative binding miRs and detection primers are also provided. (A) Comparison of candidate target mRNA levels between breast cancer samples and their normal adjacent tissue (NAT) by real-time PCR; glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. Error bars represent SD (n = 3). (B) Confirmation of target genes by real-time PCR in miR-183/-96/-182 cluster overexpression stable MCF-7 cell lines and control cells; GAPDH was used as an internal control. Error bars represent SD (n = 3). (C) Confirmation of miR-183/-96/-182 cluster targets by luciferase assay. All data were normalized to those obtained with the pGL3-Promoter vector alone. Error bars represent SD (n = 3): lower panel, sequences of miR-183 and its target sequences in the 3'-UTR of different species. (D) Protein levels of RAB21 in stable cell lines were documented by western blot with an anti-RAB21 antibody. β-Tubulin was used as the internal control; upper panel, MCF-7 cells; lower panel, T47D cells. (E) Phalloidin and 4',6-diamidino-2-phenylindole (DAPI) counterstaining results showed that the bi- and multinuclear cells were accumulated in miR-183 over-expressed MCF-7 cells; left panels, representative micrographs of single, bi- and multinuclear cells in both interphase and mitosis; right panel, quantification of bi- and multinuclear cells in different cell lines. Error bars represent SD (n = 5). Scale bars: 10 μM.