Fig 1: Identification of direct target genes of miR-193a-5p and miR-193a-3p in breast cancer cells.(a) Venn diagrams showing the numbers of target genes of miR-193a-5p and miR-193a-3p that were identified using the TargenScan tool and the microarray approach. (b) The expression levels of potential targets were analysed in breast cancer tissues in comparison with those in corresponding normal tissues from 87 breast cancer patients; these tissues were obtained from TCGA data set. (c) The proteins encoded by target genes of miR-193a-5p and miR-193a-3p were examined after transfection of MDA-MB-231 and MCF-7 cells with miR-193a-5p and miR-193a-3p mimics. (d) Schema of the luciferase constructs. The miR-193a target sequence in the 3′UTR region of target genes is shown in the upper panel and the mutant of its 3′-UTR is shown in red. Relative luciferase activity of the reporter with the 3′UTR and 3′UTR(mut) of NLN (e), CCND1 (f), and SEPN1 (g) genes was determined after cotransfection of breast cancer cells with miR-193a-5p or miR-193a-3p mimics. Firefly luciferase activity served as a normalization control.
Fig 2: The expression levels of individual target genes could alter arm expression preference of miR-193a in breast cancer cells.(a) The expression levels of miR-193a-5p were increased in breast cancer cells transfected with pmiR-193a-5p (artificial target of miR-193a-5p). (b) The expression levels of miR-193a-3p were increased in breast cancer cells transfected with pmiR-193a-3p (artificial target of miR-193a-3p). (c) The expression levels of miR-193a-5p were examined after pMIR-NLN-3′UTR and pMIR-NLN-3′UTR(mut) transfection. (d–f) The expression levels of miR-193a-3p were analyzed after pMIR-CCND1-3′UTR, pMIR-CCND1-3′UTR(mut), pMIR-PLAU-3′UTR, pMIR-PLAU-3′UTR(mut), pMIR-SEPN1-3′UTR and pMIR-SEPN1-3′UTR(mut) transfection.
Fig 3: Knockdown of the expression of direct targets of miR-193a-5p and miR-193a-3p suppressed growth and motility of breast cancer cells.(a) The expression levels of target genes were examined after transfection of MDA-MB-231 cells with siRNA. (b) The colony formation assay was performed after transfection of MDA-MB-231 cells with si-NLN, si-CCND1, si-PLAU, and si-SEPN1 for 2 wk, and a graph shows quantified values (c,d) The expression levels of target genes in MCF-7 cells were examined after siRNA transfection. (e) The colony formation assay was performed after transfection of MCF-7 cells with si-NLN, si-CCND1, and si-SEPN1 for 2 wk, and a graph shows quantified values (f–j) The migration and invasion abilities were assessed using the Transwell assay after transfection of MDA-MB-231 cells with siRNA. The cell images of a representative experiment are shown, and the graph shows values quantified using Ascent software. Data was reported as the number of colonies relative to the control (means ± SD). ***P < 0.001 vs control.
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