## Replication Study: Coding-independent regulation of the tumor suppressor PTEN by competing endogenous mRNAs <br> **Abstract:** As part of the [Reproducibility Project: Cancer Biology][1], we published a Registered Report ([Phelps et al., 2016][2]), that described how we intended to replicate selected experiments from the paper "Coding-independent regulation of the tumor suppressor PTEN by competing endogenous mRNAs" ([Tay et al., 2011][3]). Here, we report the results. We found depletion of putative PTEN competing endogenous mRNAs (ceRNAs) in DU145 cells did not impact *PTEN* 3’UTR regulation using a reporter, while the original study reported decreased activity when *SERINC1*, *VAPA*, and *CNOT6L* were depleted (Figure 3C; [Tay et al., 2011][3]). Using the same reporter, we found decreased activity when ceRNA 3’UTRs were overexpressed, while the original study reported increased activity (Figure 3D; [Tay et al., 2011][3]). 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; [Tay et al., 2011][3]); however, while the original study reported an attenuated ceRNA effect in microRNA deficient (Dicer^Ex5) HCT116 cells, we observed increased PTEN protein levels. Further, we found depletion of the ceRNAs *VAPA* or *CNOT6L* did not statistically impact DU145, wild-type HCT116, or Dicer^Ex5 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^Ex5 HCT116 cells (Figure 5B; [Tay et al., 2011][3]). 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. ---------- ### Contents **Reports:** Read the [Replication Study][4] or view the [earlier versions][5]. **Note**: 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 [here][6] or run and knit the following r markdown file and they will be downloaded automatically. To reproduce the Replication Study manuscript text run this in [R Studio][7] (note: downloads [R markdown file][8] directly from osf.io: library(httr) library(rmarkdown) GET("https://osf.io/e2cun/?action=download",write_disk("Replication_Study_24.Rmd", overwrite = T)) render("Replication_Study_24.Rmd", output_format = "word_document") <br> Also, explore the [Registered Report][9] and materials related to the Registered Report [here][10]. **Data and Material Availability:** - All associated data, protocols, analysis scripts, and other digital materials are available within this OSF project. **Experiments replicated**: Reproduce and explore the figures, analyses, data, and methods generated in this replication attempt. <br> - [*PTEN*-3'UTR luciferase reporter activity following ceRNA siRNA transfection][11] <br> -Replication of Figure 3C and Supplemental Figure S3A of Tay et al., 2011 - [*PTEN*-3'UTR luciferase reporter activity following ceRNA 3'UTR overexpression][12] <br> -Replication of Figure 3D of Tay et al., 2011 - [PTEN protein expression following ceRNA siRNA transfection][13] <br> -Replication of Figure 3G-H and Supplemental Figure S3B of Tay et al., 2011 - [Cell proliferation following ceRNA siRNA transfection][14] <br> -Replication of Figure 5B of Tay et al., 2011 **Meta-analysis**: As a measure of evaluating reproducibility a meta-analysis of each effect was performed. The forest plots, analyses, and data are available [here][15]. <br> Questions about the project can be directed to [contact+rpcb@cos.io.][16] [1]: https://osf.io/e81xl/wiki/home/ [2]: https://elifesciences.org/articles/12470 [3]: https://doi.org/10.1016/j.cell.2011.09.029 [4]: https://elifesciences.org/articles/56651 [5]: https://osf.io/n29um/ [6]: https://osf.io/e2cun/ [7]: https://www.rstudio.com [8]: https://osf.io/e2cun/ [9]: https://elifesciences.org/articles/12470 [10]: https://osf.io/c8hb5/ [11]: https://osf.io/spv4f/wiki/home/ [12]: https://osf.io/mryvq/wiki/home/ [13]: https://osf.io/drcbw/wiki/home/ [14]: https://osf.io/5c7sb/wiki/home/ [15]: https://osf.io/xgrqp/wiki/home/ [16]: mailto:contact+rpcb@cos.io