Supplementary Data from Myeloid Zinc Finger 1 Induces Migration, Invasion, and <i>In vivo</i> Metastasis through Axl Gene Expression in Solid Cancer

Supplementary Data from Myeloid Zinc Finger 1 Induces Migration, Invasion, and In vivo Metastasis through Axl Gene Expression in Solid Cance

miR-34a downregulates miR-21 by targeting the CD24/Src mediated pathway.

<p>(<b>a</b>) Western blot analysis of phosphorylated Src (p-Src), Src, CD24, phosphorylated c-jun (p-c-jun), c-Jun, c-Fos, Pdcd4 and PTEN was performed 48 h post transfection. Rko (left panel) and Geo cells (right panel) were transfected with a constitutively active Src expression construct (A-Src), empty vector (Vector), PM-34a or negative control (NC) as indicated. β-actin served as an internal control. (<b>b</b>) Luciferase reporter assays in Rko and Geo cells of the miR-21 promoter co-transfected with a constitutively active Src expression construct (A-Src), empty vector (Vector), PM-34a or negative control (NC) as indicated. Percent luciferase activity was calculated either with the miR-21 promoter or control samples set as 100%. The data are presented as the mean ± S.D. Each bar represents the mean value of three biological replicates. (<b>c</b>) miR-21 expression levels were evaluated by RT-PCR 48 h post transfection with a constitutively active Src expression construct (A-Src), empty vector (Vector), PM-34a or negative control (NC) as indicated. The data are presented as the mean ± S.D. Each bar represents the mean value of three biological replicates (p = <0.05). (<b>d</b>) Luciferase assays in Rko and Geo cells transfected with the Pdcd4-3′-UTR reporter construct together with a constitutively active Src expression construct (A-Src), empty vector (Vector), PM-34a or negative control (NC) as indicated. Percent luciferase activity was calculated either with the Pdcd4-3′-UTR or control samples set as 100%. The data are presented as the mean ± S.D. Each bar represents the mean value of three biological replicates. (<b>e</b>) The <i>in vivo</i> association of phosphorylated c-jun with the miR-21 promoter was evaluated with a ChIP assay in Rko cells after 48 h of transfection with a constitutively active Src expression construct (A-Src), empty vector (Vector), PM-34a or negative control (NC) as indicated. DNA immunoprecipitated with the p-c-jun antibody or an isotype IgG control antibody was amplified by real time PCR. Specific p-c-Jun band intensities were normalized relative to β-actin and are represented as fold change in comparison to the control.</p

miR-34a inhibits migration and invasion.

<p>(<b>a</b>) PM-34a inhibits migration <i>in vitro</i>. Rko and HCT-116 cells were transfected with a constitutively active Src expression construct (A-Src), empty vector (Vector), PM-34a or negative control (NC) as indicated. After 48 h, the cells were counted and equal numbers of cells were seeded on top of transwell plates with serum free medium and 0.1% BSA. After 14 h, the migrated cells were measured as described in Materials and Methods. Data are represented as the percentage of migrated cells, as mean±s.d. of triplicates (*p≤0.05). (<b>b</b>) PM-34a inhibits invasion <i>in vitro</i>. Rko and HCT-116 cells were transfected with a constitutively active Src expression construct (A-Src), empty vector (Vector), PM-34a or negative control (NC) as indicated. After 48 h, the cells were counted and equal numbers of cells were seeded in serum free medium and 0.1% BSA on top of transwell chambers coated with Matrigel. After 14 h, invaded cells were measured as described in Materials and Methods. Data are represented as the percentage of invaded cells, as mean±s.d. of triplicates (*p≤0.05).</p

CD24 induced miR-21 regulation is mediated through Src.

<p>(<b>a</b>) Western blot analysis of CD24, phosphorylated Src (p-Src), Src, phosphorylated c-jun (p-c-jun), c-Jun, c-Fos, Pdcd4 and PTEN was performed 48 h post transfection. Rko (left panel) and Geo cells (right panel) were transiently transfected with either an expression construct for CD24 (CD24), the empty vector (Vector), an siRNA against Src (si-Src) or a negative control siRNA (NC) as indicated. β-actin served as an internal control. (<b>b</b>) Luciferase reporter assays in Rko and Geo cells of the miR-21 promoter co-transfected with either an expression construct for CD24 (CD24), the empty vector (Vector), an siRNA against Src (si-Src) or a negative control siRNA (NC) as indicated. Percent luciferase activity was calculated either with the miR-21 promoter or control samples set as 100%. The data are presented as the mean ± S.D. Each bar represents the mean value of three biological replicates. (<b>c</b>) miR-21 expression levels were evaluated by RT-PCR 48 h post transfection with either an expression construct for CD24 (CD24), the empty vector (Vector), an siRNA against Src (si-Src) or a negative control siRNA (NC) as indicated. The data are presented as the mean ± S.D. Each bar represents the mean value of three biological replicates. (<b>d</b>) Luciferase reporter assays of the Pdcd4-3′-UTR co-transfected with either an expression construct for CD24 (CD24), the empty vector (Vector), an siRNA against Src (si-Src) or a negative control siRNA (NC) as indicated. Percent luciferase activity was calculated either with the Pdcd4-3′-UTR or control samples set as 100%. The data are presented as the mean ± S.D. Each bar represents the mean value of three biological replicates. (<b>e</b>) The <i>in vivo</i> association of phosphorylated c-jun with the miR-21 promoter was evaluated with a ChIP assay in Rko cells after 48 h of transfection with either an expression construct for CD24 (CD24), the empty vector (Vector), an siRNA against Src (si-Src) or a negative control siRNA (NC) as indicated. DNA immunoprecipitated with the p-c-jun antibody or an isotype IgG control antibody was amplified by real time PCR. Specific p-c-Jun band intensities were normalized relative to β-actin and are represented as fold change in comparison to the control.</p

CD24 Induces Expression of the Oncomir miR-21 via Src, and CD24 and Src Are Both Post-Transcriptionally Downregulated by the Tumor Suppressor miR-34a

<div><p>Cancer is a complex disease process that evolves as a consequence of multiple malfunctions in key regulatory molecular networks. Understanding these networks will be essential to combat cancer. In this study, we focussed on central players in such networks. In a series of colon and breast cancer cell lines, we found that CD24 activates Src, and induces the activation of c-Jun and expression of c-Jun and c-Fos. Thereby CD24 increases the promoter activity and expression of miR-21, which in turn suppresses expression of Pdcd4 and PTEN. Co-transfection of a CD24 expression construct and an siRNA that silences Src showed that CD24-dependent upregulation of miR-21 is mediated by Src. Additionally, we found that miR-34a post-transcriptionally downregulates CD24 and Src expression, leading to the deactivation of c-Jun, reduced expression of c-Jun and c-Fos, inhibition of miR-21, and upregulation of Pdcd4 and PTEN. Furthermore, miR-34a-mediated inhibition of Src expression reduced migration and invasion of colorectal cancer cells. Resected tumor tissues from 26 colorectal patients showed significantly lower expression of Pdcd4 and miR-34a, and higher expression of CD24, Src and miR-21 compared to the corresponding normal tissues. Moreover, CD24 positively correlated with the amount of Src protein in tumor tissues, and a trend towards an inverse correlation between miR-34a and Src protein levels was also observed. Our results reveal essential players in the complex networks that regulate the progression of solid tumors such as colorectal cancer. These findings therefore identify novel therapeutic approaches for combating tumor growth and progression.</p> </div

Supplementary Data from Src Induces <i>Urokinase Receptor</i> Gene Expression and Invasion/Intravasation via Activator Protein-1/p-c-Jun in Colorectal Cancer

Supplementary Figures S1-S2; Supplementary Table S1.</p

Endogenous expression of Pdcd4, CD24, Src, miR-21 and miR-34a in resected colorectal tissues.

<p>(<b>a</b>) Western blot analysis was performed for Pdcd4, CD24 and Src in colorectal tumors (Tumor) and corresponding normal tissues (Normal) taken from a series of 26 patients. β-Actin served as internal control. Relative mean protein amounts (Fold change comparative to normal tissue expression) of Pdcd4, CD24 and Src obtained by densitometry analysis are represented as bar graphs. Specific Pdcd4, CD24 or Src band intersities were normalized with β-actin. Pdcd4 was downregulated, CD24 and Src were upregulated significantly in the tumor tissues (p = 0.003, p = 0.05 and p = 0.001, respectively) (<b>b</b>) Real-time PCR results of miR-21 and miR-34a in the same colorectal tumor (Tumor) and normal tissue (Normal) samples. Mean relative expression (fold change compared to expression in normal tissue) of miR-21 and miR-34a is represented as bar graphs. miR-21 was upregulated and miR-34a was downregulated significantly in the tumor tissues. (p = 0.002, p = 0.05, respectively) (<b>c</b>) Lysates from 7 representative normal tissue (N) and colorectal tumor (T) samples were subjected to Western blotting and probed for the expression of Pdcd4, CD24 and Src and represented. β-Actin served as a loading control (<b>d</b>) Schematic representation of the functional network between CD24, Src, AP-1, miR-21, Pdcd4 and miR-34a.</p

Supplementary Figures 1 - 2 from Unraveling the Role of FOXQ1 in Colorectal Cancer Metastasis

PDF file - 1023K, Supplementary Figure 1: Wound healing assay in a) SW40 and b) Colo206f cell lines. Supplementary Figure 2: Evaluation of Twist 1 knockdown on migration and invasion in stable FOXQ1 over-expressing Colo206f cell lines.</p

Activated Src induces miR-21 expression.

<p>(<b>a</b>) Western blot analysis of phosphorylated Src, Src, phosphorylated c-jun, c-Jun, c-Fos, was performed 48 h post transfection. Transfection of Rko and HCT-116 cells either with vector control or with a constitutively active Src expression construct (A-Src) is shown in the left panel. Transfection of HT-29 and Geo cell lines with either negative control siRNA (NC) or an siRNA against Src (si-Src) is shown in the right panel. β-Actin served as an internal control. (<b>b</b>) Luciferase reporter assays of the miR-21 promoter co-transfected either with A-Src in Rko and HCT-116 cells (left panel) or with si-Src in HT29 and Geo cells (right panel) along with respective controls. Percent luciferase activity was calculated either with the miR-21 promoter or control samples set as 100%. The data are presented as the mean ± S.D. Each bar represents the mean value of three biological replicates (Rko: p = 0.05; HCT116: p = 0.005; HT29: p = 0.001; Geo: p<0.001). (<b>c</b>) miR-21 expression levels were evaluated by RT-PCR 48 h post transfection with A-Src in Rko and HCT-116 cells, or with si-Src in HT29 and Geo cells. The data are presented as the mean ± S.D. Each bar represents the mean value of three biological replicates (Rko: p = 0.05; HCT116: p = 0.005; HT29: p = 0.02; Geo: p = 0.02). Specific p-c-Jun band intensities were normalized relative to β-actin and are represented as fold change in comparison to the control.</p

miR-34a targets the CD24- and Src-3′-UTR and regulates their expression.

<p>(<b>a,b</b>) Luciferase assays using the CD24− and Src-3′-UTR or mutant reporter constructs transfected into HT-29 and Geo cells together with either control-miRNA (NC) or PM-34a. Percent luciferase activity was calculated either with the CD24− or Src-3′-UTR or control-miR samples set as 100%. The data are presented as the mean ± S.D. Each bar represents the mean value of three technical replicates. (<b>c</b>) Geo, Rko, HT-29 and MDA-MB-231 cells were transfected either with control miRNA or PM-34a, and 48 h later protein was isolated and western blot analysis for CD24 and Src was performed (left panel). Rko and MDA-MB-231 cells were transfected either with control miRNA or AM-34a, and 48 h later protein was isolated and western blot analysis for CD24 and Src was performed (right panel).</p