The WAVE2 modulation of integrin activities is mediated through the regulation of miR-29 miRNA. A, Volcano plot from the RNA-seq analysis of the differentially expressed genes between CTRL and W2-KO MDA-MB-231 cells. The X-axis represents the log2 fold change in expression levels and the Y-axis shows the P values. Red dots: upregulated genes; Blue dots: downregulated genes; Black dots: no significant change. The dots of the genes of interest are labeled. B, Bars and values representation of the log2 fold change in expression levels of miR-29a and miR-29b derived from the RNA-seq data. C, Quantification of miR-29a (left) and miR-29b (right) expression levels from CTRL and W2KO MDA-MB-231 cells using qRT-PCR. Values are plotted as fold change to the CTRL cells, after normalization to U6 expression levels. D, Nucleotide sequence and location of the seed sequence of miR-29 in the 3′-UTR of ITGB1 mRNA. The sequence alignment with the miR-29 sequence is also shown. E, Representative WB analysis of protein lysates from CTRL and miR-29–expressing MDA-MB-231 probed with anti ITGB1 antibody. β-Actin was used for loading control. F, Quantification of miR-29a (left) and miR-29b (right) expression levels from CTRL, W2KO, or miR-29–expressing MDA-MB-231 cells using qRT-PCR. Values are plotted as fold change to the CTRL cells, after normalization to U6 expression levels. G, Quantification of luciferase activity of ITGB1–3′-UTR. Firefly luciferase reporter plasmid pmirGlo, empty (EV), containing the 3′-UTR of ITGB1 with wild-type (miR-29) or scrambled miR-29 seed sequence (SCRAM) was transiently transfected into HEK293 along with a miR-29–expressing vector. Luciferase activities were measured after 48 hours and plotted after being normalized Renilla luciferase. Representative histograms of flow cytometry analyses for cell surface expression of ITGB1 (H) and activity: HUTS4 binding (I) of CTRL, W2-KO, and ITGB1-KO or miR-29–expressing MDA-MB-231 cells. Shift to the right indicates increased expression or activity. Data are the means ± SD (n = 3; **, P < 0.01; Student t test). Data shown are representative of three replicates.
