PMC:7572125 / 7392-13730 JSONTXT

{"project":"2_test","denotations":[{"id":"33073769-22000013-27525615","span":{"begin":464,"end":468},"obj":"22000013"},{"id":"33073769-26943900-27525616","span":{"begin":538,"end":542},"obj":"26943900"},{"id":"33073769-22000013-27525617","span":{"begin":1807,"end":1811},"obj":"22000013"},{"id":"33073769-22000013-27525618","span":{"begin":2548,"end":2552},"obj":"22000013"}],"text":"ceRNA depletion on PTEN-3’UTR luciferase reporter activity\nWe independently replicated an experiment to test if putative PTEN ceRNAs modulate the 3’UTR of PTEN. This experiment used a chimeric luciferase construct tagged with the PTEN 3’UTR (Luc-PTEN-3’UTR) to uncouple regulation of PTEN via 3’UTR-targeting microRNAs from PTEN mRNA transcription and protein stability. This is similar to what was reported in Figure 3C and Supplemental Figure S3A of Tay et al., 2011 and described in Protocol 1 in the Registered Report (Phelps et al., 2016). DU145 cells were co-transfected with Luc-PTEN-3’UTR and siRNAs targeting the same putative PTEN ceRNAs as the original study. Knockdown efficiency was examined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The average reduction in gene expression relative to control siRNA was 65% when SERINC1, VAPA, CNOT6L, or PTEN were targeted, but was only 21% for ZNF460 (Figure 1—figure supplement 1C), despite transfection efficiency being at least 90% based on a fluorescent transfection indicator (Figure 1—figure supplement 1A). Luciferase activity was decreased in PTEN depleted cells (average RLU = 12%) relative to control siRNA (average RLU = 100%); however, luciferase activity when the putative PTEN ceRNAs were targeted for depletion were similar to control siRNA (Figure 1, Figure 1—figure supplement 1B). All planned comparisons were not statistically significant (see Figure 1 legend). The original study reported statistically significant decreased luciferase activity with siRNA-mediated depletion of SERINC1 (average RLU = 70%), VAPA (average RLU = 48%), CNOT6L (average RLU = 70%), or PTEN (average RLU = 20%), but not for knockdown of ZNF460 (average RLU = 109%), compared to control siRNA (average RLU = 100%) (Tay et al., 2011). The range of luciferase values reported in the original study had relative standard deviations (RSDs) (control = 9%; SERINC1 = 10%; VAPA = 6%; CNOT6L = 5%; ZNF460 = 8%; PTEN = 5%) that were much smaller than the RSDs observed in this replication attempt (control = 36%; SERINC1 = 57%; VAPA = 36%; CNOT6L = 50%; ZNF460 = 33%; PTEN = 19%), which is one of the factors that could influence if statistical significance is reached, particularly since the sample size of this replication attempt was determined a priori to detect the effect based on the originally reported data. The original study also reported an achieved knockdown of 90% or greater when SERINC1, VAPA, CNOT6L, or PTEN were targeted, but was 65% for ZNF460 (Tay et al., 2011). The difference in achieved knockdown between the original study and this replication attempt is a possible reason for the differences in Luc-PTEN-3’UTR outcomes. A higher level of knockdown might be required to observe an effect with this experimental design. Although, unlike experiments that evaluate protein function where a higher level of knockdown or a longer period of time is usually needed to observe a phenotype (Curtis and Nardulli, 2009; O’Keefe, 2013), the putative ceRNA function of these mRNAs should correspond to the level of knockdown. Thus, a 65% knockdown would have been expected to capture ~72% of the effect observed in the original study that reported a 90% knockdown. To summarize, for this experiment, we found results that were not statistically significant where predicted, varied in direction relative to the original study for the putative PTEN ceRNAs, and in the same direction as the original study for cells transfected with siPTEN.\nFigure 1. Luciferase activity in DU145 cells co-transfected with siRNA against PTEN ceRNAs and a luciferase-PTEN 3’UTR reporter construct.\nDU145 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\u003e0.99; siNC and siZNF: t(6) = 0.899, uncorrected p=0.403, Bonferroni corrected p\u003e0.99; siNC and siVAPA: t(6) = 0.225, uncorrected p=0.829, Bonferroni corrected p\u003e0.99; siNC and siCNO: t(6) = 0.426, uncorrected p=0.685, Bonferroni corrected p\u003e0.99; 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/.\nFigure 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.\nThis 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 \u003e90%. (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/."}

{"project":"MyTest","denotations":[{"id":"33073769-22000013-27525615","span":{"begin":464,"end":469},"obj":"22000013"},{"id":"33073769-26943900-27525616","span":{"begin":538,"end":542},"obj":"26943900"},{"id":"33073769-22000013-27525617","span":{"begin":1807,"end":1811},"obj":"22000013"},{"id":"33073769-22000013-27525618","span":{"begin":2548,"end":2552},"obj":"22000013"}],"namespaces":[{"prefix":"_base","uri":"https://www.uniprot.org/uniprot/testbase"},{"prefix":"UniProtKB","uri":"https://www.uniprot.org/uniprot/"},{"prefix":"uniprot","uri":"https://www.uniprot.org/uniprotkb/"}],"text":"ceRNA depletion on PTEN-3’UTR luciferase reporter activity\nWe independently replicated an experiment to test if putative PTEN ceRNAs modulate the 3’UTR of PTEN. This experiment used a chimeric luciferase construct tagged with the PTEN 3’UTR (Luc-PTEN-3’UTR) to uncouple regulation of PTEN via 3’UTR-targeting microRNAs from PTEN mRNA transcription and protein stability. This is similar to what was reported in Figure 3C and Supplemental Figure S3A of Tay et al., 2011 and described in Protocol 1 in the Registered Report (Phelps et al., 2016). DU145 cells were co-transfected with Luc-PTEN-3’UTR and siRNAs targeting the same putative PTEN ceRNAs as the original study. Knockdown efficiency was examined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The average reduction in gene expression relative to control siRNA was 65% when SERINC1, VAPA, CNOT6L, or PTEN were targeted, but was only 21% for ZNF460 (Figure 1—figure supplement 1C), despite transfection efficiency being at least 90% based on a fluorescent transfection indicator (Figure 1—figure supplement 1A). Luciferase activity was decreased in PTEN depleted cells (average RLU = 12%) relative to control siRNA (average RLU = 100%); however, luciferase activity when the putative PTEN ceRNAs were targeted for depletion were similar to control siRNA (Figure 1, Figure 1—figure supplement 1B). All planned comparisons were not statistically significant (see Figure 1 legend). The original study reported statistically significant decreased luciferase activity with siRNA-mediated depletion of SERINC1 (average RLU = 70%), VAPA (average RLU = 48%), CNOT6L (average RLU = 70%), or PTEN (average RLU = 20%), but not for knockdown of ZNF460 (average RLU = 109%), compared to control siRNA (average RLU = 100%) (Tay et al., 2011). The range of luciferase values reported in the original study had relative standard deviations (RSDs) (control = 9%; SERINC1 = 10%; VAPA = 6%; CNOT6L = 5%; ZNF460 = 8%; PTEN = 5%) that were much smaller than the RSDs observed in this replication attempt (control = 36%; SERINC1 = 57%; VAPA = 36%; CNOT6L = 50%; ZNF460 = 33%; PTEN = 19%), which is one of the factors that could influence if statistical significance is reached, particularly since the sample size of this replication attempt was determined a priori to detect the effect based on the originally reported data. The original study also reported an achieved knockdown of 90% or greater when SERINC1, VAPA, CNOT6L, or PTEN were targeted, but was 65% for ZNF460 (Tay et al., 2011). The difference in achieved knockdown between the original study and this replication attempt is a possible reason for the differences in Luc-PTEN-3’UTR outcomes. A higher level of knockdown might be required to observe an effect with this experimental design. Although, unlike experiments that evaluate protein function where a higher level of knockdown or a longer period of time is usually needed to observe a phenotype (Curtis and Nardulli, 2009; O’Keefe, 2013), the putative ceRNA function of these mRNAs should correspond to the level of knockdown. Thus, a 65% knockdown would have been expected to capture ~72% of the effect observed in the original study that reported a 90% knockdown. To summarize, for this experiment, we found results that were not statistically significant where predicted, varied in direction relative to the original study for the putative PTEN ceRNAs, and in the same direction as the original study for cells transfected with siPTEN.\nFigure 1. Luciferase activity in DU145 cells co-transfected with siRNA against PTEN ceRNAs and a luciferase-PTEN 3’UTR reporter construct.\nDU145 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\u003e0.99; siNC and siZNF: t(6) = 0.899, uncorrected p=0.403, Bonferroni corrected p\u003e0.99; siNC and siVAPA: t(6) = 0.225, uncorrected p=0.829, Bonferroni corrected p\u003e0.99; siNC and siCNO: t(6) = 0.426, uncorrected p=0.685, Bonferroni corrected p\u003e0.99; 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/.\nFigure 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.\nThis 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 \u003e90%. (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/."}