PMC

(A) Diagrammatic representation of miR-632 binding site. Solid black boxes represent exons.
(B) Diagrammatic representation of binding of miR-632 seed sequence to its target site in DNAJB6.
(C) Cross species sequence comparison miR-632. Highlighted boxes with bold italicized underlined letters depict deviation from human miR-632 sequence. mfe = minimum-free energy.

Total RNA from 15 breast tumors was analyzed by qRT-PCR for miR-632 and DNAJB6 levels from. The levels were compared with average of normal levels as calibrator, designated as 1. The graph is presented in logarithmic scale for Y-axis.

(A) (I) MCF10AT cells were transiently transfected miR-632-pIRES2EGFP and were allowed to invade through matrigel coated filters for 18 hrs. Invaded cells were visualized using crystal violet, and the cell number was counted and compared to invasion of cells transfected with empty vector control. (II) MCF10AT cells were transiently transfected with empty pIRES2EGFP vector or miR-632-pIRES2EGFP and their growth in 3-D was assessed. Arrows point the invasive outgrowth. Experiments were performed in triplicate and the experiment was performed two times.
(B) (I) MDA-MB-231 cells were treated with X-miR-632 (100 nM) or the control and the invasion assay was performed as described before. (II) MDA-MB-231 cells were transfected with miRNA inhibitor scrambled control clone pEZX-AM01 (control) or anti-miR-632 pIRES2EGFP and their growth in 3-D was assessed. Arrows point the invasive outgrowth.. Experiments were performed in triplicate and the experiment was performed two times.

(A) Levels of each of the miRNAs, miR-197, miR-424 and miR-632 from human Breast cancer cell lines MCF10AT, MCF10CA1d.cl.1, SUM159 and MDA-MB-231 were compared to immortalized human mammary epithelial cell line MCF10A using real time-quantitive-PCR. The graph is presented in logarithmic scale for Y-axis. Reactions were performed in triplicate and the experiment was performed twice.
(B) mir-424 and miR-632 were transiently expressed in MCF10A. Total protein extract (20μg) was resolved using SDS-PAGE and subjected to western blot analysis for DNAJB6 levels. β-actin level was determined to confirm equal loading.

(A) pMIR-REPORT-DNAJB6 (containing putative binding site of miR-632 from the ORF of DNAJB6) was co-transfected with miR-632 expression vector, miR-632-pIRES2EGFP or empty vector control. The assay was performed in triplicate and the experiment was performed twice. The luciferase activity readings were normalized with activity from a co-transfected β-gal expressing control. The error bars represent standard error of mean (SEM)
(B) Levels of DNAJB6 and PTCH1 transcript were evaluated from MCF10A cells treated with miR-632-pIRES2EGFP or empty vector control. GAPDH was used as endorse control. The error bars represent standard error of mean (SEM). The reactions were performed in triplicate and the experiment was performed three times.
(C) MDA-MB-231 cells were treated with X-miR-632 or control (50 and 100 nM). Total protein extract (30μg) was resolved using SDS-PAGE and subjected to western blot analysis for DNAJB6 levels. β-actin levels were determined as loading control.