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Replication Study: Coding-independent regulation of the tumor suppressor PTEN by competing endogenous mRNAs

  1. Hongyan Wang
  2. Hanna S Radomska
  3. Mitch A Phelps
  4. Reproducibility Project: Cancer Biology  Is a corresponding author
  1. Pharmacoanalytic Shared Resource (PhASR), Comprehensive Cancer Center, The Ohio State University, United States
  2. Science Exchange, United States
  3. Center for Open Science, United States
Replication Study
Cite this article as: eLife 2020;9:e56651 doi: 10.7554/eLife.56651
5 figures, 1 table and 1 additional file

Figures

Figure 1 with 1 supplement
Luciferase activity in DU145 cells co-transfected with siRNA against PTEN ceRNAs and a luciferase-PTEN 3’UTR reporter construct.

DU145 cells were transfected with a luciferase reporter with a fragment of the 3’UTR of PTEN. Cells were also co-transfected with non-targeting control siRNA (siNC) or siRNA plasmids targeting SERINC1 (siSER), ZNF460 (siZNF), VAPA (siVAPA), CNOT6L (siCNO), or PTEN (siPTEN). Cells were harvested 72 hr later for luciferase activity. Relative luminescence unit (RLU) is presented for each condition relative to the siNC condition. Means reported and error bars represent SD from four independent biological repeats. Two-sample t-test of RLU values between siNC and siSER: t(6) = 0.177, uncorrected p=0.866 with a priori Bonferroni adjusted significance threshold of 0.01, Bonferroni corrected p>0.99; siNC and siZNF: t(6) = 0.899, uncorrected p=0.403, Bonferroni corrected p>0.99; siNC and siVAPA: t(6) = 0.225, uncorrected p=0.829, Bonferroni corrected p>0.99; siNC and siCNO: t(6) = 0.426, uncorrected p=0.685, Bonferroni corrected p>0.99; Wilcoxon-Mann-Whitney test of RLU values between siNC and siPTEN: U = 16, uncorrected p=0.029, Bonferroni corrected p=0.143. Additional details for this experiment can be found at https://osf.io/spv4f/.

Figure 1—figure supplement 1
Knockdown efficiency and individual repeats of luciferase-PTEN 3’UTR reporter assay in DU145 cells co-transfected with siRNA against PTEN ceRNAs.

This is the same experiment as Figure 1. (A) Representative microscopy images (10X magnification) of DY-547-labeled siGLO RISC-Free control transfected DU145 cells 48 hr after transfection. Transfection efficiency was estimated to be >90%. (B) Independent biological repeats of luciferase reporter assay. Relative luminescence unit (RLU) is presented for each condition relative to the siNC condition. Means reported and error bars represent SD from three technical replicates. (C) Independent biological repeats of RT-qPCR analysis. Expression of each transcript after transfection of its respective siRNA relative to negative control transfection (siNC) is presented. Transcripts listed on y-axis. Means reported and error bars represent SD from three technical replicates. One-sample t-tests of transcript expression data after transfection of respective siRNA to a constant of 1 (relative value of siNC). SERINC1: t(3) = 45.8, uncorrected p=2.30×10−5, Bonferroni corrected p=1.15×10−4; ZNF460: t(3) = 2.98, uncorrected p=0.059, Bonferroni corrected p=0.294; VAPA: t(3) = 8.94, uncorrected p=0.0030, Bonferroni corrected p=0.015; CNOT6L: t(3) = 11.3, uncorrected p=0.0015, Bonferroni corrected p=0.0074; PTEN: t(3) = 11.2, uncorrected p=0.0015, Bonferroni corrected p=0.0077. Additional details for this experiment can be found at https://osf.io/spv4f/.

Figure 2 with 1 supplement
Luciferase activity in DU145 cells co-transfected with 3’UTR of PTEN ceRNAs and a luciferase-PTEN 3’UTR reporter construct.

DU145 cells were transfected with a luciferase reporter with a fragment of the 3’UTR of PTEN. Cells were also co-transfected with empty vector (EV) or plasmids that express the 3’UTR of SERINC1 (SER 3’U), VAPA (VAPA 3’U1 and VAPA 3’U2), CNOT6L (CNOT 3’U1 and CNOT 3’U2), or PTEN (PTEN 3’U). Cells were harvested 72 hr later for luciferase activity. Relative luminescence unit (RLU) is presented for each condition relative to the EV condition. Means reported and error bars represent SD from six independent biological repeats. Two-sample t-test of RLU values between SER 3’U and EV: t(10) = 3.32, uncorrected p=0.0077 with a priori Bonferroni adjusted significance threshold of 0.0083, Bonferroni corrected p=0.046; VAPA 3’U1 and EV: t(10) = 3.13, uncorrected p=0.011, Bonferroni corrected p=0.064; VAPA 3’U2 and EV: t(10) = 4.83, uncorrected p=6.90×10−4, Bonferroni corrected p=0.0041; CNOT 3’U1 and EV: t(10) = 4.42, uncorrected p=0.0013, Bonferroni corrected p=0.0078; CNOT 3’U2 and EV: t(7.1) = 5.09, uncorrected p=0.0014, Bonferroni corrected p=0.0082; PTEN 3’U and EV: t(5.6) = 7.50, uncorrected p=3.99×10−4, Bonferroni corrected p=0.0024. Additional details for this experiment can be found at https://osf.io/mryvq/.

Figure 2—figure supplement 1
Individual repeats of luciferase-PTEN 3’UTR reporter assay in DU145 cells co-transfected with 3’UTR of PTEN ceRNAs.

This is the same experiment as Figure 2. Independent biological repeats of luciferase reporter assay. Relative luminescence unit (RLU) is presented for each condition relative to the EV condition. Means reported and error bars represent SD from three technical replicates. Additional details for this experiment can be found at https://osf.io/mryvq/.

Figure 3 with 1 supplement
PTEN protein expression in wild-type and DICER mutant HCT116 cells depleted of PTEN ceRNAs.

Wild-type (WT) and DICER mutant (DicerEx5) HCT116 cells were transfected with non-targeting control siRNA (siNC) or siRNA plasmids targeting SERINC1 (siSER), VAPA (siVAPA), CNOT6L (siCNO), or PTEN (siPTEN). Cells were harvested 72 hr later for Western blot analysis. (A) Relative protein expression (PTEN/HSP90) are presented for each condition. Western blot bands were quantified, PTEN levels were normalized to HSP90, with protein expression presented relative to siNC. Means reported and error bars represent SD from three independent biological repeats for wild-type HCT116 cells and four repeats for DicerEx5 HCT116 cells. Analysis of wild-type HCT116 cells: one-way ANOVA (equal variance) on PTEN/HSP90 expression: F(4,10) = 25.4, I=3.18×10−5. Planned contrasts between siNC and siSER: t(10) = 1.94, uncorrected I=0.082 with a priori Bonferroni adjusted significance threshold of 0.0125, Bonferroni corrected p=0.326; siNC and siVAPA: t(10) = 5.44, uncorrected p=2.85×10−4, Bonferroni corrected p=0.0011; siNC and siCNOT: t(10) = 3.69, uncorrected p=0.0042, Bonferroni corrected p=0.017; siNC and siPTEN: t(10) = 9.34, uncorrected p=2.97×10−6, Bonferroni corrected p=1.19×10−5. Analysis of DicerEx5 HCT116 cells: one-way ANOVA (unequal variance) on PTEN/HSP90 expression: F(4,6.0) = 19.3, p=0.0014. Planned comparisons: siNC and siSER: two-sample t-test, t(6) = 3.96, uncorrected p=0.0074 with a priori Bonferroni adjusted significance threshold of 0.0125, Bonferroni corrected p=0.030; siNC and siVAPA: two-sample t-test, t(6) = 0.896, uncorrected p=0.405, Bonferroni corrected p>0.99; siNC and siCNOT: Welch’s t-test, t(4.36) = 2.92, uncorrected p=0.039, Bonferroni corrected p=0.156; siNC and siPTEN: two-sample t-test, t(6) = 4.15, uncorrected p=0.0060, Bonferroni corrected p=0.024. (B) Representative Western blots probed with an anti-PTEN antibody and anti-HSP90 antibody. Additional details for this experiment can be found at https://osf.io/drcbw/.

Figure 3—figure supplement 1
Knockdown efficiency and individual repeats of PTEN protein expression in wild-type and DICER mutant HCT116 cells transfected with siRNA against PTEN ceRNAs.

This is the same experiment as Figure 1. (A) Independent biological repeats of Western blot assay. PTEN/HSP90 protein expression is presented for each condition relative to the siNC condition. (B) Independent biological repeats of RT-qPCR analysis. Expression of each transcript after transfection of its respective siRNA relative to negative control transfection (siNC) is presented. Transcripts listed on y-axis. Means reported and error bars represent SD from three technical replicates. One-sample t-tests of transcript expression data after transfection of respective siRNA to a constant of 1 (relative value of siNC). Wild-type HCT116 cells: SERINC1: t(2) = 8.41, uncorrected p=0.014, Bonferroni corrected p=0.055; VAPA: t(2) = 120, uncorrected p=6.98×10−5, Bonferroni corrected p=2.79×10−4; CNOT6L: t(2) = 10.4, uncorrected p=0.0091, Bonferroni corrected p=0.036; PTEN: t(2) = 30.6, uncorrected p=0.0011, Bonferroni corrected p=0.0043. DicerEx5 HCT116 cells: SERINC1: t(2) = 28.5, uncorrected p=0.0012, Bonferroni corrected p=0.0049; VAPA: t(2) = 48.3, uncorrected p=4.28×10−4, Bonferroni corrected p=0.0017; CNOT6L: t(2) = 3.23, uncorrected p=0.084, Bonferroni corrected p=0.336; PTEN: t(2) = 242, uncorrected p=1.71×10−5, Bonferroni corrected p=6.83×10−5. Additional details for this experiment can be found at https://osf.io/drcbw/.

Figure 4 with 1 supplement
Growth of cells depleted of PTEN ceRNAs.

DU145, wild-type (WT) and DICER mutant (DicerEx5) HCT116 cells were transfected with either a non-targeting control siRNA (siNC) or siRNA plasmids targeting VAPA (siVAPA), CNOT6L (siCNO), or PTEN (siPTEN). Crystal violet proliferation assays were performed each day as indicated starting the day after transfection. Relative OD590 was calculated relative to the average Day 0 values for each condition. Means reported and error bars represent SD from five independent biological repeats for DU145 cells and four times for HCT116 WT and DicerEx5 cells. Analysis on the area under the curve (AUC) for each condition of each biological repeat (reported as dot plot in Figure 4—figure supplement 1A). Analysis results for DU145 cells: one-way ANOVA (equal variance): F(3,16) = 3.27, p=0.049. Planned contrasts between siNC and siVAPA: t(16) = 0.648, uncorrected p=0.526 with a priori Bonferroni adjusted significance threshold of 0.0167, Bonferroni corrected p>0.99; siNC and siCNOT6L: t(16) = 0.950, uncorrected p=0.356, Bonferroni corrected p>0.99; siNC and siPTEN: t(16) = 2.09, uncorrected p=0.053, Bonferroni corrected p=0.158. Analysis of HCT116 cells: two-way ANOVA interaction between DICER status (wild-type or Ex5) and siRNA target: F(3,24) = 0.734, p=0.542; main effect of DICER status: F(1,24) = 1.81, p=0.191; main effect of siRNA target: F(3,24) = 12.1, p=5.20×10−5. Planned contrasts in HCT116 WT cells: siNC and siVAPA: t(24) = 2.02, uncorrected p=0.054 with a priori Bonferroni adjusted significance threshold of 0.0083, Bonferroni corrected p=0.325; siNC and siCNOT6L: t(24) = 0.506, uncorrected p=0.618, Bonferroni corrected p>0.99; siNC and siPTEN: t(24) = 3.03, uncorrected p=0.0057, Bonferroni corrected p=0.034. Planned contrasts in HCT116 DICEREx5 cells: siPTEN and siVAPA: t(24) = 2.43, uncorrected p=0.023, Bonferroni corrected p=0.138; siPTEN and siCNOT6L: t(24) = 4.57, uncorrected p=1.25×10−4, Bonferroni corrected p=7.48×10−4; siNC and siPTEN: t(24) = 4.31, uncorrected p=2.42×10−4, Bonferroni corrected p=0.0015. Additional details for this experiment can be found at https://osf.io/5c7sb/.

Figure 4—figure supplement 1
Knockdown efficiency and individual repeats of cell growth assay in cells transfected with siRNA against PTEN ceRNAs.

This is the same experiment as Figure 5. (A) Independent biological repeats of cell growth assay. Dot plot with means reported as crossbars and error bars represent SD. (B) Independent biological repeats of RT-qPCR analysis. Expression of each transcript after transfection of its respective siRNA relative to negative control transfection (siNC) is presented. Transcripts listed on y-axis. Means reported and error bars represent SD from three technical replicates. One-sample t-tests of transcript expression data after transfection of respective siRNA to a constant of 1 (relative value of siNC). DU145 cells: VAPA: t(4) = 58.6, uncorrected p=5.09×10−7, Bonferroni corrected p=1.53×10−6; CNOT6L: t(4) = 36.3, uncorrected p=3.42×10−6, Bonferroni corrected p=1.03×10−5; PTEN: t(4) = 9.05, uncorrected p=8.27×10−4, Bonferroni corrected p=0.0025. Wild-type HCT116 cells: VAPA: t(3) = 18.6, uncorrected p=3.40×10−4, Bonferroni corrected p=0.0010; CNOT6L: t(3) = 108, uncorrected p=1.73×10−6, Bonferroni corrected p=5.19×10−6; PTEN: t(3) = 28.1, uncorrected p=9.90×10−5, Bonferroni corrected p=2.97×10−4. DicerEx5 HCT116 cells: VAPA: t(3) = 14.8, uncorrected p=6.73×10−4, Bonferroni corrected p=0.0020; CNOT6L: t(3) = 27.9, uncorrected p=1.02×10−4, Bonferroni corrected p=3.05×10−4; PTEN: t(3) = 178, uncorrected p=3.89×10−7, Bonferroni corrected p=1.17×10−6. Additional details for this experiment can be found at https://osf.io/5c7sb/.

Meta-analyses of each effect.

Effect size and 95% confidence interval are presented for Tay et al., 2011, this replication study (RP:CB), and a random effects meta-analysis of those two effects. For each effect, Cohen’s d or Glass’ delta, which are standardized differences between the two indicated measurements, is reported. Sample sizes used in Tay et al., 2011 and RP:CB are reported under the study name. (A) These effects are related to the change in luciferase activity between the conditions reported in Figure 1 of this study and Figure 3C of Tay et al., 2011. Meta-analysis p values: siNC and siSER (p=0.374); siNC and siZNF (p=0.233); siNC and siVAPA (p=0.316); siNC and siCNO (p=0.253); siNC and siPTEN (p=0.079). (B) These effects are related to the change in luciferase activity between the conditions reported in Figure 2 of this study and Figure 3D of Tay et al., 2011. Meta-analysis p values: SER 3’U and EV (p=0.881); VAPA 3’U1 and EV (p=0.624); VAPA 3’U2 and EV (p=0.754); CNO 3’U1 and EV (p=0.790); CNO 3’U2 and EV (p=0.716); PTEN 3’U and EV (p=0.766). (C) These effects are related to the differences in PTEN protein expression between the conditions reported in Figure 3 of this study and Figure 3H of Tay et al., 2011. Meta-analysis p values: WT HCT116: siNC and siSER (p=0.091); siNC and siVAPA (p=2.99×10−7); siNC and siCNO (p=0.049); siNC and siPTEN (p=0.0041): DicerEx5 HCT116: siNC and siSER (p=2.78×10−4); siNC and siVAPA (p=0.215); siNC and siCNO (p=3.70×10−4); siNC and siPTEN (p=0.229). (D) These effects are related to the differences in cell growth between the conditions reported in Figure 4 of this study and Figure 5B of Tay et al., 2011. Meta-analysis p values: DU145: siVAPA and siNC (p=0.255); siCNO and siNC (p=0.554); siPTEN and siNC (p=0.082): WT HCT116: siVAPA and siNC (p=0.045); siCNO and siNC (p=0.287); siPTEN and siNC (p=0.082): DicerEx5 HCT116: siVAPA and siPTEN (p=0.075); siCNO and siPTEN (p=0.001); siPTEN and siNC (p=0.048). Additional details for these meta-analyses can be found at https://osf.io/xgrqp/.

Tables

Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional
information
Cell line (Homo sapiens, male)DU145ATCCcat#:HTB-81; RRID:CVCL_0105
Cell line (H. sapiens, male)Wild-type HCT116 cellsHorizon Discoverycat# HD R02-019; RRID:CVCL_HD76
Cell line (H. sapiens, male)DICEREx5 HCT116 cellsHorizon Discoverycat# HD R02-019; RRID:CVCL_HD76
Recombinant DNA reagentpsiCHECK-2+PTEN3’UTRAddgeneplasmid# 50936; RRID:Addgene_50936
Recombinant DNA reagentSERINC1 3’UTRdoi:10.1016/j.cell.2011.09.029Shared by Dr. Pier Paolo Pandolfi, Beth Israel Deaconess Medical Center
Recombinant DNA reagentVAPA 3’UTR1doi:10.1016/j.cell.2011.09.029Shared by Dr. Pier Paolo Pandolfi, Beth Israel Deaconess Medical Center
Recombinant DNA reagentVAPA 3’UTR2doi:10.1016/j.cell.2011.09.029Shared by Dr. Pier Paolo Pandolfi, Beth Israel Deaconess Medical Center
Recombinant DNA reagentCNOT6L 3’UTR1doi:10.1016/j.cell.2011.09.029Shared by Dr. Pier Paolo Pandolfi, Beth Israel Deaconess Medical Center
Recombinant DNA reagentCNOT6L 3’UTR2doi:10.1016/j.cell.2011.09.029Shared by Dr. Pier Paolo Pandolfi, Beth Israel Deaconess Medical Center
Recombinant DNA reagentPTEN 3’UTRdoi:10.1016/j.cell.2011.09.029Shared by Dr. Pier Paolo Pandolfi, Beth Israel Deaconess Medical Center
Sequence-based reagentsiGlo RISC-free siRNADharmaconcat#:D-001600–01
Sequence-based reagentsiGENOME non-targeting siRNADharmaconcat#:D-001210–02
Sequence-based reagentsiGENOME SERINC1Dharmaconcat# M-010725–00
Sequence-based reagentsiGENOME ZNF460Dharmaconcat# M-032012–01
Sequence-based reagentsiGENOME VAPADharmaconcat# M-021382–01
Sequence-based reagentsiGENOME CNOT6LDharmaconcat# M-016411–01
Sequence-based reagentsiGENOME PTENDharmaconM-003023–02
Sequence-based reagentTaqMan probe SERINC1Thermo Fisher ScientificHs00380375_m1
Sequence-based reagentTaqMan probe ZNF460Thermo Fisher ScientificHs01104252_m1
Sequence-based reagentTaqMan probe VAPAThermo Fisher ScientificHs00427749_m1
Sequence-based reagentTaqMan probe CNOT6LThermo Fisher ScientificHs00375913_m1
Sequence-based reagentTaqMan probe PTENThermo Fisher ScientificHs02621230_s1
Sequence-based reagentTaqMan probe PARD3Thermo Fisher ScientificHs00969077_m1
Antibodyrabbit anti-PTENCell Signaling Technologycat#:9559; clone:138G5; RRID:AB_3908101:1000 dilution
Antibodymouse anti-HSP90BD Biosciencescat#:610419; clone:68; RRID:AB_3977981:1000 dilution
AntibodyHRP-conjugated donkey anti-rabbitGE Healthcarecat#:NA934; RRID:AB_7722061:2000 dilution
AntibodyHRP-conjugated rabbit anti-mouseAbcamcat#:ab6728; RRID:AB_9554401:2000 dilution
Software, algorithmVeritas Microplate Luminometer softwareTurner BioSystemspart#:998–9100; RRID:SCR_018534
Software, algorithmStepOne Plus Real-Time PCR softwareApplied BiosystemsRRID:SCR_014281Version 2.3
Software, algorithmImageJdoi:10.1038/nmeth.2089RRID:SCR_003070Version 1.50a
Software, algorithmGen5 softwareBioTek InstrumentsRRID:SCR_017317Version 2.05.5
Software, algorithmR Project for statistical computinghttps://www.r-project.orgRRID:SCR_001905Version 3.5.1

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