Journal: Molecular Systems Biology

Article Title: A genome‐scale screen reveals context‐dependent ovarian cancer sensitivity to mi RNA overexpression

doi: 10.15252/msb.20156308

Figure Lengend Snippet: Transfection conditions were optimized using normalized cell viability values upon transfection of mimic negative control and a pan‐toxic siRNA, siUBB. Values plotted as the mean of three separate experiments with the bars representing the range. For each microRNA mimic, a standard deviation value was calculated for mean viability across the panel of 16 cell lines (blue curve). In addition, a within‐miRNA seed family standard deviation value was calculated for mean viability across the cell panel (red curve). Lastly, a within‐replicate standard deviation value was calculated (black curve). A kernel density estimation was fit to each of the three standard deviation distributions and plotted. The indicated microRNA mimics were clustered, using hierarchical APC, based on their viability z ‐scores across 16 cell lines. A Euclidean distance metric was used. Node colors indicate cluster membership. A high‐resolution searchable and zoomable pdf has been provided as to facilitate visualization of cluster entities. The indicated ovarian cancer cell lines were clustered, using hierarchical APC, according to miR mimic viability z ‐scores (400 miRs). Node colors indicate cluster membership. RNASeq data for the indicated cell lines (acquired from the CCLE or internal analysis (this study)) was first filtered to select the top 20% of the most highly variant genes (2,686 genes total). The cell lines were then clustered using hierarchical APC based on a Euclidean distance metric. Node colors indicate cluster membership. Nodes in the gene expression‐based APC from (E) were relabeled according to their cluster membership in the miR mimic response‐based clusters from (D).

Article Snippet: A total of 9.8 μl of serum free medium containing 0.2 μl of RNAiMAX transfection reagent was then added to each well using a TiterTek multidrop (except for PEO4 where 0.2 μl of DharmaFECT 3 was used) followed by a 20‐ to 30‐min incubation at room temperature.

Techniques: Transfection, Negative Control, Standard Deviation, Variant Assay, Gene Expression

Journal: Molecular Systems Biology

Article Title: A genome‐scale screen reveals context‐dependent ovarian cancer sensitivity to mi RNA overexpression

doi: 10.15252/msb.20156308

Figure Lengend Snippet: A Tukey plot of the range of z ‐scores for all mimics screened in each cell line screened. Z ‐scores are representative of the mean viability of 3 replicates. Quantitative PCR (qPCR) analysis of endogenous miR‐146a expression revealed no association between level of expression in a cell line and toxicity in the screen (top panel). To facilitate line‐to‐line comparison, endogenous expression values were normalized to a reference cell line (Hey cells). qPCR showed similar levels of overexpression among all cell lines (bottom panel). Bars in red display the endogenous expression of miR‐146a (normalized to RNU6B using the comparative CT method), while bars in black display the overexpression of miR‐146a mimic after transfection (normalized similar to endogenous expression). qPCR analysis of endogenous miR‐505 expression revealed no association between level of expression in a cell line and toxicity in the screen (top panel). To facilitate line‐to‐line comparison, endogenous expression values were normalized to a reference cell line (Hey cells). qPCR showed similar levels of overexpression among all cell lines (bottom panel). Endogenous and overexpression values displayed as in (B). Bars represent the mean of 2 replicates ± SD. Kaplan–Meier plots of training and validation sets of tumors using miRNA expression from both Agilent arrays and Illumina miRseq showed that higher expression of miR‐505 correlated with increased patient survival. Kaplan–Meier plots of training and validation sets of tumors using miRNA expression from both Agilent arrays and Illumina miRseq showed that higher expression of miR‐146a correlated with increased patient survival.

Article Snippet: A total of 9.8 μl of serum free medium containing 0.2 μl of RNAiMAX transfection reagent was then added to each well using a TiterTek multidrop (except for PEO4 where 0.2 μl of DharmaFECT 3 was used) followed by a 20‐ to 30‐min incubation at room temperature.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Comparison, Over Expression, Transfection, Biomarker Discovery

Journal: Molecular Systems Biology

Article Title: A genome‐scale screen reveals context‐dependent ovarian cancer sensitivity to mi RNA overexpression

doi: 10.15252/msb.20156308

Figure Lengend Snippet: The consequence of miR‐155 and miR‐181b mimics on cell viability, 5 days post‐transfection, relative to a negative control miRNA mimic is shown across a panel of normal and ovarian cancer cell lines as indicated. Each data point is the mean of n = 3/cell line. In the screen, miR‐155 was found to reduce cell viability ( z ‐score < −2) in one cell line, and TCGA expression data revealed no significant change in expression of this miRNA in ovarian tumors. miR‐181 was found to reduce viability ( z ‐score < −2) in 2 cell lines, and TCGA expression data revealed no significant change in expression of this miRNA in ovarian tumors. N, normal cell lines (IHH, HOSE, HBEC3, HBEC13, HBEC30, and HBEC34); EOC, epithelial ovarian cancer cell lines. miR‐155 data points for each cell line are shown in black, while miR‐181b data points are displayed in red. Immunoblots indicate suppression of serum‐induced AKT phosphorylation at S473 and T308 in response to miR‐155 and suppression of T308 phosphorylation in response to miR‐181b. miR‐155 and miR‐181b reduced cell viability in GDC0941‐sensitive breast cancer cell lines. Error bars indicate mean ± SD ( n = 3). Differential localization of E‐cadherin in PEO1 and PEO4 cells 48 h post‐transfection with the indicated oligos. Cells were counterstained with phalloidin and DAPI. Stimulation of PEO4 cells with 10 ng/ml TGFβ1 significantly sensitized the cells to AKT inhibition with AKT Inhibitor X. Each point represents the mean of 3 experiments ± SD and * denotes P ‐value < 0.05 by Student's t ‐test. Cell viability upon expression of miR‐155 or miR‐181a mimics in 41 NSCLC cell lines and 5 human bronchial epithelial cell lines. Each data point is the mean of n = 3/cell line. R 2 from Pearson correlation. P ‐value calculated from Student's t ‐distribution. Source data are available online for this figure.

Article Snippet: A total of 9.8 μl of serum free medium containing 0.2 μl of RNAiMAX transfection reagent was then added to each well using a TiterTek multidrop (except for PEO4 where 0.2 μl of DharmaFECT 3 was used) followed by a 20‐ to 30‐min incubation at room temperature.

Techniques: Transfection, Negative Control, Expressing, Western Blot, Phospho-proteomics, Inhibition

Journal: Molecular Systems Biology

Article Title: A genome‐scale screen reveals context‐dependent ovarian cancer sensitivity to mi RNA overexpression

doi: 10.15252/msb.20156308

Figure Lengend Snippet: Consequence of miR‐124 on EOC cell viability (as in Fig 2A). N, normal cell lines (IHH and HOSE). In the screen, miR‐124 was found to significantly reduce cell viability ( z ‐score < −2) in 6 cell lines, and TCGA expression data revealed no significant change in expression of this miRNA in ovarian tumors. miR‐124 induced expression of neuronal marker proteins Tuj1 (TUBB3) and MAP2 in ES2 cells 48 h post‐transfection. Cells were counterstained with DAPI and phalloidin. miR‐124‐responsive genes in PEO1 cells were enriched for TargetScan predicted targets. P ‐value from hypergeometric distribution. Source data are available online for this figure.

Article Snippet: A total of 9.8 μl of serum free medium containing 0.2 μl of RNAiMAX transfection reagent was then added to each well using a TiterTek multidrop (except for PEO4 where 0.2 μl of DharmaFECT 3 was used) followed by a 20‐ to 30‐min incubation at room temperature.

Techniques: Expressing, Marker, Transfection

Journal: Molecular Systems Biology

Article Title: A genome‐scale screen reveals context‐dependent ovarian cancer sensitivity to mi RNA overexpression

doi: 10.15252/msb.20156308

Figure Lengend Snippet: Expression of miR‐124 mimic reduced expression of PTBP1 (PTB) and CTDSP1 (SCP1) but not other members of the REST complex on a microarray. Each bar represents the log 2 of the ratio of the means of three experiments of mir‐124 mimic expression to mimic negative control expression of a single probe on the microarray. miR‐124 mimic expression reduced expression of mesenchymal markers ITGB1, VIM, and TWIST2 and increased expression of neuronal markers CDH2, VAMP2, and MAP2 according to the microarray data. Each bar represents the log 2 of the ratio of the means of three experiments of mir‐124 mimic expression to mimic negative control expression of a single probe on the microarray. SIX4, EYA1, EYA2, EYA3, and EYA4 are predicted targets of miR‐124 in TargetScan ( www.targetscan.org ). SIX4 expression is significantly enriched in human ovarian tumors relative to normal ovarian tissue. P ‐value from Welch two‐sample t ‐test. N, unmatched normal ovarian tissue; P, primary epithelial ovarian tumors; R, recurrent epithelial ovarian tumors. Knockdown of SIX4 reduced cell viability similar to expression of a miR‐124 mimic in PEO1 cells (mean ± SD of 3 experiments). Expression of a SIX4 construct that cannot be targeted by miR‐124 partially rescues toxicity due to miR‐124 over expression in PEO1 cells. Mean ± SD of 6 experiments. Two‐way ANOVA analysis, *** P < 0.005. SIX4 knockdown did not induce activation of caspase‐3 and caspase‐7. Depletion of SIX4 reduced BrdU incorporation in ES2 cells and resulted in decreased Hoechst‐positive nuclei 48 h post‐transfection. SIX4 knockdown also resulted in decreased cells in S‐phase as seen in PI‐stained ES2 cells 48 h post‐transfection. All bars represent the mean ± SD of three experiments. Knockdown of SIX4 reduced expression of multiple cyclin proteins in PEO1 and PEO4 cells on the microarray. Each bar represents the log 2 of the ratio of the means of three experiments of SIX4 siRNA transfection to siRNA negative control transfection of a single probe on the microarray. SIX4 knockdown induced expression of STRADB in PEO1 and PEO4 cells according to the microarray data. Each bar represents the log 2 of the ratio of the means of three experiments of SIX4 siRNA transfection to siRNA negative control transfection of a single probe on the microarray. Schematic of predicted interactions of miR‐124 with cell cycle and cell differentiation machinery. Source data are available online for this figure

Article Snippet: A total of 9.8 μl of serum free medium containing 0.2 μl of RNAiMAX transfection reagent was then added to each well using a TiterTek multidrop (except for PEO4 where 0.2 μl of DharmaFECT 3 was used) followed by a 20‐ to 30‐min incubation at room temperature.

Techniques: Expressing, Microarray, Negative Control, Knockdown, Construct, Over Expression, Activation Assay, BrdU Incorporation Assay, Transfection, Staining, Cell Differentiation

Journal: Molecular Systems Biology

Article Title: A genome‐scale screen reveals context‐dependent ovarian cancer sensitivity to mi RNA overexpression

doi: 10.15252/msb.20156308

Figure Lengend Snippet: Immunoblots indicate miR‐124‐induced SIX4 depletion. siSIX4 is shown as an antibody control. Consequence of SIX4 depletion on EOC cell viability (as in Fig 2A, N, normal cell lines: HOSE, HBEC30). In vivo knockdown of SIX4 using neutral liposome‐incorporated siRNA reduced tumor burden and ascites volume in an orthotopic xenograft mouse model. Box‐and‐whisker plot of tumor weight (left panel) or ascites volume (right panel) from n = 7 or 8 mice per condition as indicated. *P ‐value from Student's t ‐test. Consequence of SIX4 depletion in PEO1 cells on E‐cadherin plasma membrane accumulation 48 h after transfection. Cells were counterstained with phalloidin and DAPI. Immunoblots indicate consequence of SIX4 depletion on LKB1 accumulation and AMPK pathway activation. Source data are available online for this figure.

Article Snippet: A total of 9.8 μl of serum free medium containing 0.2 μl of RNAiMAX transfection reagent was then added to each well using a TiterTek multidrop (except for PEO4 where 0.2 μl of DharmaFECT 3 was used) followed by a 20‐ to 30‐min incubation at room temperature.

Techniques: Western Blot, Control, In Vivo, Knockdown, Whisker Assay, Clinical Proteomics, Membrane, Transfection, Activation Assay

Journal: Bio-protocol

Article Title: RNA Interference Screening to Identify Proliferation Determinants in Breast Cancer Cells

doi: 10.21769/BioProtoc.2435

Figure Lengend Snippet: Recipe of diluted lipid To account for pipetting loss, 187.5 μl (12.5 x 15 μl) diluted lipid will be made in Opti -MEM.

Article Snippet: The Cybio program ( ) will pipette 10 μl from 0.24 μM siRNA ER library plate in V-bottom plate, then mixed into Costar 96-well plate that already has 10.5 μl of diluted RNAimax transfection reagent.

Techniques: Blocking Assay

Journal: Bio-protocol

Article Title: RNA Interference Screening to Identify Proliferation Determinants in Breast Cancer Cells

doi: 10.21769/BioProtoc.2435

Figure Lengend Snippet: Efficiency of a variety of transfection reagents was tested in MCF7 cells.

Article Snippet: The Cybio program ( ) will pipette 10 μl from 0.24 μM siRNA ER library plate in V-bottom plate, then mixed into Costar 96-well plate that already has 10.5 μl of diluted RNAimax transfection reagent.

Techniques: Transfection