Figure 5

Sub-clones of SW620 cells isolated from lung metastasis show TWIST1-dependnent over-expression of miR-372/373. (a) Outline of sub-clone isolation. (b) Morphology of the control cell lines (panels 1–3) and three metastasis-derived sub-clones of V/SW620-Luc (panels 4–6) stained with anti-vimentin antibodies plus DAPI. (c) Relative levels of mature form (upper panels) or the primary transcripts (lower panels) of miR-15b/16 (left), miR-21 (center), and miR-372/373 (right), as determined by qRT–PCR and agarose gel electrophoresis of RT–PCR products, respectively. (d) Immunoblot detection of E-cadherin, vimentin, and TWIST1. The sample numbering is the same as in B and C. (e) Structure of the promoter region of the miR-372/373 cluster. Three miRNAs, miR-371, miR-372, and mir-373 (blue boxes) are expressed as a single primary transcript (Houbaviy et al., 2005) (red box). There are four predicted TWIST1 binding motifs (that is, E-boxes) (black bars) near the transcriptional start site (arrow): three in the up-stream region and one down-stream of the start site. (f) Effects of TWIST1 on miR-372/373 expression. SW620 cells were stably transfected with either a vacant expression vector (V) or the vector expressing either of the two TWIST1 cDNA clones, BC033434 (#1) or BC083139 (#2). Both encode an identical protein; the only difference is that #2 has a longer 5′UTR. Cell lysates prepared from transfected and untransfected (−) cells were subjected to immunoblot assay to monitor TWIST1 expression (Control: α-tubulin) (Top panels). Total RNAs was subjected to qRT–PCR to assess the levels of mature miR-372/373 (bar graph) or to RT–PCR followed by agarose gel electrophoresis (control: GAPDH) to visualize the primary transcript harboring miR-372/373 (bottom panels). (g) Chromatin immunoprecipitation assay for TWIST binding sites. Nuclear lysates of C15 cells were precipitated using no (−), non-specific (N; rabbit IgG), or anti-TWIST1 (T) antibodies, and the DNA associated with the precipitates was used to amplify a region of approximately 120 bp containing each E-box (see Methods in SI for the primers used). (h) Luciferase reporter assay with deletion mutants of the miR-372/373 promoter. SW620 cells were co-transfected with a TWIST1-expression vector and a promoter-luciferase reporter construct containing either all four E-boxes (bars 2, 3) or only 2 (bars 5, 6) or 3 E-boxes (bars 7, 8). Luciferase activity was determined 48 h after transfection (n=3). *P<0.05.