ERG transcription factor is required to maintain high <i>TDRD1</i> expression.

<p>(A) <i>ERG</i> and <i>TDRD1</i> mRNA expression levels in VCaP cells measured 72 h after gene silencing with siRNAs. Three independent experiments were performed in triplicate. (B) ERG and TDRD1 protein expression in VCaP cells 72 h after gene silencing with siRNAs.</p

<i>TDRD1</i> is co-expressed with <i>ERG</i> but not with <i>ETV1</i> in prostate cancer.

<p>(A) Correlation analysis of mRNA levels measured by qRT-PCR in <i>TMPRSS2:ERG</i>-negative (ERG-, n = 30) and <i>TMPRSS2:ERG</i>-positive (ERG+, n = 17) prostate cancers as well as adjacent benign prostate tissue (n = 46). Pearson correlation coefficient is shown. (B) Analysis of mRNA expression in prostate cell lines by qRT-PCR. Two independent experiments were performed in triplicate. Human testis RNA was used as positive control for <i>TDRD1</i> expression. (C) Analysis of protein expression in prostate cell lines by western blotting. (D) Analysis of mRNA expression in hematopoietic cancer cell lines by qRT-PCR. Two independent experiments were performed in triplicate.</p

<i>TDRD1</i> promoter associated CpG island is hypomethylated in <i>TMPRSS2:ERG</i>-positive prostate cancer.

<p>(A) Analysis of <i>TDRD1</i> promoter methylation in prostate tumors by MeDIP-Seq. The values represent the average degree of DNA methylation of the 500-bp bins. (B) Correlation analysis of <i>TDRD1</i> promoter methylation and <i>TDRD1</i> mRNA expression in prostate cancer. The Spearman correlation coefficient is shown.</p

<i>TDRD1</i> does not control LINE1 activity in <i>TMPRSS2:ERG</i>-positive VCaP cells.

<p>(A) mRNA expression analysis of <i>PIWIL</i> genes in prostate cell lines by qRT-PCR. Testis RNA was used as a positive control. (B) mRNA expression analysis of LINE1 ORF2 in VCaP cells following 5 days of treatment with 5-aza-2′-deoxycytidine. (C) mRNA expression analysis of LINE1 ORF2 in VCaP cells following prolonged (8 days) <i>ERG</i> or <i>TDRD1</i> silencing. (D) Metabolic viability assay of VCaP cells treated with siRNAs. One (B), two (A) or three (C, D) independent experiments were performed in triplicate.</p

ERG-induced loss of epigenetic repression at the <i>TDRD1</i> promoter is a major mechanism of <i>TDRD1</i> overexpression.

<p>(A) DNA methylation analysis of the <i>TDRD1</i> promoter-associated CpG island in prostate cell lines by bisulfite sequencing. Average methylation level of the whole CpG island calculated from five sequenced colonies is shown (%). (B) Analysis of mRNA expression in LNCaP cells after treatment with the demethylating agent 5-aza-2′-deoxycytidine. Insert: analysis of protein expression in LNCaP cells after treatment with 5-aza-2′-deoxycytidine. 75µg of protein lysate from LNCaP cells was used per lane. (C) Analysis of mRNA expression in stable LNCaP clones overexpressing <i>ERG</i>. Two independent experiments were performed in triplicate. Insert: ERG expression analysis at 48 h in LNCaP clones by western blotting. (D) Bisulfite sequencing of the <i>TDRD1</i> promoter-associated CpG island in LNCaP cells 48 h after induction of ERG expression with doxycycline. (E) Bisulfite sequencing of the <i>TDRD1</i> promoter-associated CpG island 96 h after silencing of <i>ERG</i> in VCaP cells. The data shown in (D) and (E) are mean % of methylation of the entire CpG island calculated from 11–12 sequenced clones.</p

Differential expression of miRNAs in colon tumor and metastasis tissues.

<p>(<b>A</b>) Top 25 up- and down-regulated miRNAs comparing tumor (left) or metastasis (right) tissues versus normal colon samples as analyzed by Illumina sequencing. All depicted miRNAs sufficed a p-value threshold ≤0.05. A star indicates samples with p≤0.01. (<b>B</b>) Venn diagram of microRNAs expressed in colorectal cancer patients, as determined by Illumina sequencing. (Left) Numbers of detected miRNAs, specific for each tissue (normal (N) = 19, tumor (T) = 34, metastases (M) = 29) and in all tissues (559). (Middle) Venn diagram of the significantly up-regulated miRNAs (p-value ≤0.05) for all comparisons (N/T, N/M and T/M). (Right) Venn diagram of the significantly down-regulated miRNAs (p-value ≤0.05) for all comparisons (N/T, N/M and T/M).</p

Functional assays on miRNA-1 as a potential tumor-suppressor gene.

<p>(<b>A</b>) AlamarBlue cell viability assay to test the effect of miRNA-1. SW480 (primary colon cancer cell line) and SW620 (corresponding metastases cell line) cells were transfected with miRNA-1 mimics (+miR-1) or mock transfected (−miR-1) and measured using an spectrophotometer after 24 h, 48 h and 72 h. The miRNA-1 level was determined by TaqMan assays for mature microRNAs. (<b>B</b>) “Wound healing” assay for miRNA-1 in SW480 and SW620 cells. After 24 h of transfection with miRNA-1 mimics a uniform scratch was generated through each confluent cell layer and “wound” closure was documented after 24 h using a phase-contrast microscope (n = 2). (<b>C</b>) AlamarBlue cell viability assay in SW480 and SW620 cells after camptothecin treatment alone or in combination with miRNA-1. Cell viability was measured after 0 h, 24 h and 48 h of drug treatment using a spectrophotometer.</p

Clinical parameters of the colorectal cancer patients.

a<p>microsatellite.</p