The most differentially expressed miRNAs in stage II colorectal adenocarcinomas and their ability to induce phenotypic changes in the high-throughput analysis.

1<p>A FC _(log2)≤−1.50 and ≥1.50 and a p-value≤0.01 was considered significant (Mann-Whitney U test).</p><p>NA: miRNAs not included in the pre-miRNA library from Ambion.</p><p>+: miRNAs that induced phenotypic changes (Top-40 ranked).</p><p>(+): miRNAs that induced phenotypic changes in at least one cell line (not Top-40 ranked).</p><p>A: Induction of apoptosis.</p><p>P: Inhibition of proliferation.</p

mRNA profiling of HCT116 cells upon ectopic expression of miR-375 and miR-375 target identification.

<p>(A) Reconstitution of mature miR-375 upon transfection with pre-miR-375 or Scr (RT-qPCR). (B) Cumulative fraction plotted as a function of log2 fold changes. The mRNAs were dichotomized according to the presence or absence of minimum one seed match in the 3′UTR or according to target prediction using Target Scan v5.2. The mRNAs with minimum one 7mer-m8 seed match within the 3′ UTR showed a higher propensity to down-regulation upon miR-375 over-expression. (C) The mRNAs were ranked according to fold change and grouped into a total of 23 bins. Upper panel: The average 7mer-8m seed frequency within the 3′UTR regions in each bin was calculated. Bottom panel: Overall miRNA-induced mRNA fold change. (D and E) Relative expression of HELLS, NOLC1, YAP1, BIRC5 and BCL2L1 upon ectopic miR-375 expression using RT-qPCR. (F) Western blots demonstrating the effect of miR-375 on the protein level of HELLS, NOLC1 and YAP1 in HCT116 cells. Loading control: β-actin. *<i>p</i><0.05. (G–H) Ago 2 immunoprecipitation. (G) RT-qPCR expression analysis of YAP1 in the cell lysates of miR-375 or Scr transfected cells (input) used for Ago2 immunoprecipitation. (H) Ago2 immunoprecipitation from cell lysates of miR-375 or Scr transfected cells (IP) followed by YAP1 expression analysis using RT-qPCR. Immunoprecipitation with a FLAG antibody was used as negative control. A 1∶1 ratio of the lysates from miR-375 and Scr transfected cells was used for FLAG immunoprecipitation. The columns represent the mean of 3 replicates ± sd.</p

Generation and characterization of stable HCT116 cells with inducible miR-375 expression (HCT116-miR-375H).

<p>(A) Dox treatment induces increased expression of miR-375 in HCT116_miR-375H cells. The Relative expression of miR-375 was measured using RT-qPCR. The columns represent the mean of 3 replicates ± sd.*p-value<0.05 when compared to HCT116_ScrH cells. (B) xCELLigence real-time monitoring of cell proliferation. The cell index from time 12–96 hours is shown. Dox was added at time 0. (C) Dox treatment significantly reduces the proliferation of HCT116_miR-375H cells. Following the real-time monitoring in B, the slope (rate of changes in cell index) was calculated from time 60–80 hours (i.e. when changes in cell viability were apparent) and presented graphically. (D) Dox treatment specifically induces Caspase 3/7 dependent apoptosis in HCT116 miR-375H cells. The Caspase 3/7 activity was examined by fluorometric kinetic analysis and expressed relative to the Caspase 3/7 activity in untreated HCT116_ScrH cells. Z-DEVD-fmk (DEVD) was added to the cells six hours post-transfection. Data are presented as ±sd. of at least 2 independent experiments each with three biological replicates. *p-value<0.05. (E) Western blotting demonstrating down-regulation of YAP1 in dox treated HCT116_miR-375H cells compared to non-treated and HCT116_ScrH cells. Loading control: β-actin. (F) miR-375 expression reduces tumor growth <i>in vivo</i>. Growth curves of tumors generated in nude mice injected with HCT116_miR-375H cells treated with (n = 4) or without (n = 4) dox in the drinking water. Dox was added to the drinking water when the tumor size was >50 mm³. Data marks and bars represent the mean ±sd. *p-value<0.05.</p

Model of the role of miR-375 in the regulation of apoptotic death.

<p>Model of the role of miR-375 in the regulation of apoptotic death.</p

siRNA knock-down of YAP1 reduces both mRNA and protein expression in HCT116 cells.

<p>(A) The relative expression of YAP1, BCL2L1 and BIRC5 mRNA in cells transfected with YAP1 siRNA_1 or siRNA_2. The columns represent the mean of 3 replicates ± sd. *p-value<0.05 when compared to Scr. (B) Western blot demonstrating the effect of YAP1 knock-down at the protein level. Loading control: β-actin. (C) The effect of YAP1 knock-down on the viability CRC cells (MTT assay). Data are presented as ±sd. of at least 3 independent experiments each with three biological replicates and normalized to Scr. *p-value<0.05 and MTT reduction >20%. (D) Cellular death (LDH release assay): The cellular death was expressed as percentage of released LDH out of total cellular LDH. At least two independent experiments were carried out and performed in triplicates. The result of one representative experiments ±sd. is shown. *p-value<0.05. (E) Induction of apoptosis (Caspase 3/7 activity): The Caspase 3/7 activity in the lysate of siRNA transfected cells was examined by fluorometric kinetic analysis and expressed relative to the Caspase 3/7 activity in “Scr” transfected cells. Z-DEVD-fmk (DEVD) was added to the cells six hours post-transfection. Data are presented as ±sd. of at least 2 independent experiments each with three biological replicates. *p-value<0.05.</p

Expression of known direct miR-375 targets.

Δ<p>Analyzed in 24 normal mucosa and 30 MSS adenocarcinomas that has previously been profiled using Human Exon 1.0 ST arrays (Thorsen K et al. Alternative Splicing of SLC39A14 in Colorectal Cancer is Regulated by the Wnt Pathway,</p><p>Molecular and Cellular Proteomics, 2011).</p><p>ND: not detected (median log intensity <7).</p><p>NS: not significant (a p-value≤0.05 was considered significant).</p>‡<p>: not present on the Human Gene 1.0ST.</p><p>*Hs: Homo sapiens.</p>¤<p>Mm: Mus musculus.</p>∧<p>The probes on the Human Gene 1.0ST arrays recognize more than one transcript.</p

Phenotypic analyses of selected miRNAs in HCT116 cells upon ectopic expression of the miRNAs.

<p>(A) Cellular viability (MTT assay): Data are presented as ±sd. of at least 3 independent experiments each with three biological replicates and normalized to Scr. *p-value<0.05 and MTT reduction >20%. (B) Cellular death (LDH release assay): The cellular death was expressed as percentage of released LDH out of total cellular LDH. At least two independent experiments were carried out and performed in triplicates. The result of one representative experiments ±sd. is shown. *p-value<0.05. (C) Induction of apoptosis (Caspase 3/7 activity): The Caspase 3/7 activity in the lysate of pre-miRNA transfected cells was examined by fluorometric kinetic analysis and expressed relative to the Caspase 3/7 activity in “Scr” transfected cells. Data are presented as ±sd. of at least 2 independent experiments each with three biological replicates. *p-value<0.05. (D) Inhibition of miR-375 induced apoptosis by the Caspase 3/7 inhibitor z-DEVD-fmk. The Caspase 3/7 activity was measured as described in (C). Z-DEVD-fmk (DEVD) (25 µM) was added to the cells six hours post-transfection. Non treated cells (NT), Lipofectamine only (Lipo) and pre-miR miRNA Precursor Molecules-Negative Control #1 (Scr) were included as negative controls in all assays. The pre-miR-145 transfected cells were included as a positive control for performance of the MTT assay. (E) The down-regulation of miR-375 is a result of reduced expression in the epithelial cells of the tumor. Expression of miR-375 in laser capture microdissected colorectal cancer tissue. The expression was analyzed in epithelial and stromal cells from paired colorectal adenocarcinomas (n = 3) and adjacent normal (n = 3) colon mucosa LCM biopsies using RT-qPCR. The columns represent the mean expression in three samples ± sd.</p

Individual knock-down of HELLS and NOLC1 reduces both mRNA and protein expression in HCT116 cells.

<p>(A) The relative expression of HELLS and NOLC1 mRNA in cells transfected with siRNA_1 and siRNA_2. The columns represent the mean of 3 replicates ± sd. (B) Western blots demonstrating the effect of siRNA target knock-down at the protein level. Loading control: β-actin. (C) The effect of HELLS and NOLC1 siRNA_1 on the viability CRC cells. Data are presented as ±sd. of at least 3 independent experiments each with three biological replicates and normalized to Scr. *p-value<0.05 and MTT reduction >20%. (D) Cellular death (LDH release assay): The cellular death was expressed as percentage of released LDH out of total cellular LDH. At least two independent experiments were carried out and performed in triplicates. The result of one representative experiments ±sd. is shown. *p-value<0.05. (E) Induction of apoptosis (Caspase 3/7 activity): The Caspase 3/7 activity in the lysate of siRNA transfected cells was examined by fluorometric kinetic analysis and expressed relative to the Caspase 3/7 activity in “Scr” transfected cells. Data are presented as ±sd. of at least 2 independent experiments each with three biological replicates. *p-value<0.05. A Scr siRNA was included as negative controls in all assays.</p

Methylation of <i>MIR-375 in</i> CRC cell lines and clinical samples using Infinium HumanMethylation450 BeadChips.

<p>(A and C) Methylation of <i>MIR-375</i> in 8 CRC cell lines (A) and 12 normal colon mucosa samples paired with colorectal adenomas (n = 3) or adenocarcinomas (n = 9) (C). CpG sites in close proximity to <i>MIR-375</i> (CpG1-11) were analyzed. (B and D) Expression analysis of miR-375 in the CRC cells lines (B) and the paired clinical samples (RT-qPCR) (D). The miR-375 expression was measured in triplicates and normalized to miR-340. * indicate the pairs of clinical samples with significant miR-375 down-regulation (FC_(log2)>1.5). N: normal colon mucosa, A: adenoma and C: adenocarcinoma.</p

miRNAs differentially expressed in normal colon mucosa versus stage II CRC.

<p>Hierarchical clustering of miRNAs with a significant different expression between colorectal adenocarcinomas and normal colon mucosa samples (p≤0.01). The rows represent individual miRNAs and the columns represent individual tissue samples. The scale represents the intensity of the gene expression (log2 scale ranging between −2.98 and 2.98) (p-values and FCs are found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096767#pone-0096767-t002" target="_blank">Table 2</a>). The miRNAs marked with blue were found to inhibit proliferation and/or induce apoptosis in the high-throughput screen (Top-40 ranked miRNAs).</p