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<h2>Replication Study: Coding-independent regulation of the tumor suppressor PTEN by competing endogenous mRNAs</h2> <p><br> <strong>Abstract:</strong></p> <p>As part of the <a href="https://osf.io/e81xl/wiki/home/" rel="nofollow">Reproducibility Project: Cancer Biology</a>, we published a Registered Report (<a href="https://elifesciences.org/articles/12470" rel="nofollow">Phelps et al., 2016</a>), that described how we intended to replicate selected experiments from the paper "Coding-independent regulation of the tumor suppressor PTEN by competing endogenous mRNAs" (<a href="https://doi.org/10.1016/j.cell.2011.09.029" rel="nofollow">Tay et al., 2011</a>). Here, we report the results. We found depletion of putative PTEN competing endogenous mRNAs (ceRNAs) in DU145 cells did not impact <em>PTEN</em> 3’UTR regulation using a reporter, while the original study reported decreased activity when <em>SERINC1</em>, <em>VAPA</em>, and <em>CNOT6L</em> were depleted (Figure 3C; <a href="https://doi.org/10.1016/j.cell.2011.09.029" rel="nofollow">Tay et al., 2011</a>). Using the same reporter, we found decreased activity when ceRNA 3’UTRs were overexpressed, while the original study reported increased activity (Figure 3D; <a href="https://doi.org/10.1016/j.cell.2011.09.029" rel="nofollow">Tay et al., 2011</a>). In HCT116 cells, ceRNA depletion resulted in decreased PTEN protein levels, a result similar to the findings reported in the original study (Figure 3G,H; <a href="https://doi.org/10.1016/j.cell.2011.09.029" rel="nofollow">Tay et al., 2011</a>); however, while the original study reported an attenuated ceRNA effect in microRNA deficient (Dicer<sup>Ex5</sup>) HCT116 cells, we observed increased PTEN protein levels. Further, we found depletion of the ceRNAs <em>VAPA</em> or <em>CNOT6L</em> did not statistically impact DU145, wild-type HCT116, or Dicer<sup>Ex5</sup> HCT116 cell proliferation. The original study reported increased DU145 and wild-type HCT116 cell proliferation when these ceRNAs were depleted, which was attenuated in the Dicer<sup>Ex5</sup> HCT116 cells (Figure 5B; <a href="https://doi.org/10.1016/j.cell.2011.09.029" rel="nofollow">Tay et al., 2011</a>). Differences between the original study and this replication attempt, such as variance between biological repeats, are factors that might have influenced the results. Finally, we report meta-analyses for each result.</p> <hr> <h3>Contents</h3> <p><strong>Reports:</strong> Read the <a href="https://elifesciences.org/articles/56651" rel="nofollow">Replication Study</a> or view the <a href="https://osf.io/n29um/" rel="nofollow">earlier versions</a>.</p> <p><strong>Note</strong>: In order to successfully run and knit the Replication Study Manuscript R Markdown file, you must install a series of necessary R packages. You can review the necessary packages included in the checkpoint manifest in the markdown file <a href="https://osf.io/e2cun/" rel="nofollow">here</a> or run and knit the following r markdown file and they will be downloaded automatically.</p> <p>To reproduce the Replication Study manuscript text run this in <a href="https://www.rstudio.com" rel="nofollow">R Studio</a> (note: downloads <a href="https://osf.io/e2cun/" rel="nofollow">R markdown file</a> directly from <a href="http://osf.io" rel="nofollow">osf.io</a>:</p> <pre class="highlight"><code>library(httr) library(rmarkdown) GET(&quot;<a href="https://osf.io/e2cun/?action=download" rel="nofollow">https://osf.io/e2cun/?action=download</a>&quot;,write_disk(&quot;Replication_Study_24.Rmd&quot;, overwrite = T)) render(&quot;Replication_Study_24.Rmd&quot;, output_format = &quot;word_document&quot;)</code></pre> <p><br> Also, explore the <a href="https://elifesciences.org/articles/12470" rel="nofollow">Registered Report</a> and materials related to the Registered Report <a href="https://osf.io/c8hb5/" rel="nofollow">here</a>.</p> <p><strong>Data and Material Availability:</strong> - All associated data, protocols, analysis scripts, and other digital materials are available within this OSF project.</p> <p><strong>Experiments replicated</strong>: Reproduce and explore the figures, analyses, data, and methods generated in this replication attempt. <br> - <a href="https://osf.io/spv4f/wiki/home/" rel="nofollow"><em>PTEN</em>-3'UTR luciferase reporter activity following ceRNA siRNA transfection</a> <br> -Replication of Figure 3C and Supplemental Figure S3A of Tay et al., 2011</p> <ul> <li><a href="https://osf.io/mryvq/wiki/home/" rel="nofollow"><em>PTEN</em>-3'UTR luciferase reporter activity following ceRNA 3'UTR overexpression</a> <br> -Replication of Figure 3D of Tay et al., 2011</li> <li><a href="https://osf.io/drcbw/wiki/home/" rel="nofollow">PTEN protein expression following ceRNA siRNA transfection</a> <br> -Replication of Figure 3G-H and Supplemental Figure S3B of Tay et al., 2011 </li> <li><a href="https://osf.io/5c7sb/wiki/home/" rel="nofollow">Cell proliferation following ceRNA siRNA transfection</a> <br> -Replication of Figure 5B of Tay et al., 2011</li> </ul> <p><strong>Meta-analysis</strong>: As a measure of evaluating reproducibility a meta-analysis of each effect was performed. The forest plots, analyses, and data are available <a href="https://osf.io/xgrqp/wiki/home/" rel="nofollow">here</a>. <br></p> <p>Questions about the project can be directed to <a href="mailto:contact+rpcb@cos.io">contact+rpcb@cos.io.</a></p>
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