## Replication Study: The microRNA miR-34a inhibits prostate cancer stem cells and metastasis by directly repressing CD44 <br> **Abstract:** In 2015, as part of the [Reproducibility Project: Cancer Biology][1], we published a Registered Report ([Jia Li et al., 2015][2]), that described how we intended to replicate selected experiments from the paper “The microRNA miR-34a inhibits prostate cancer stem cells and metastasis by directly repressing CD44" ([Liu et al., 2011][3]). Here we report the results. We found the microRNA, miR-34a, was expressed at twice the level in CD44+ prostate cancer cells purified from xenograft tumors (LAPC4 cells) compared to CD44- LAPC4 cells, whereas the original study reported miR-34a was underexpressed in CD44+ LAPC4 cells (Figure 1B; [Liu et al., 2011][4]). When LAPC4 cells engineered to express miR-34a were injected into mice, we did not observe changes in tumor growth or CD44 expression; however, unexpectedly miR-34a expression was lost in vivo. In the original study, LAPC4 cells expressing miR-34a had a statistically significant reduction in tumor regeneration and reduced CD44 expression compared to control (Figures 4A and Supplemental Figures 4A,B and 5C; [Liu et al., 2011][5]). Furthermore, when we tested if miR-34a regulated CD44 through binding sites in the 3’UTR we did not find a statistically significant difference, whereas the original study reported miR-34a decreased CD44 expression that was partially abrogated by mutation of the binding sites in the CD44 3’UTR (Figure 4D; [Liu et al., 2011][6]). Finally, where possible, we report meta-analyses for each result. ---------- ### Contents **Reports:** Read the [Replication Study][7], or view the [preprint versions][8]. To reproduce the Replication Study manuscript text run this in [R Studio][9] (note: downloads [R Markdown file][10] directly from osf.io): library(httr) library(rmarkdown) GET("https://osf.io/e89mt/?action=download",write_disk("Replication_Study_28.Rmd", overwrite = T)) render("Replication_Study_28.Rmd", output_format = "word_document") Also, explore the [Registered Report][11] and materials related to the Registered Report [here][12]. <br> **Data and Material Availability:** All associated data, protocols, analysis scripts, and other digital materials are available within this OSF project. Flow cytometry data for this study has also been deposited at Flow Repository, where it is directly accessible at https://flowrepository.org/id/FR-FCM-ZYNB. <br> **Experiments replicated:** Reproduce and explore the figures, analyses, data, and methods generated in this replication attempt. - [Protocols 1, 2, & 3: Expression of miR-34a in LAPC4 sub-populations][13] - Protocol 1, as outlined in the Registered Report, describes the maintenance of the LAPC4 xenograft tumor tissue in NOD/SCID mice. - Protocol 2, as outlined in the Registered Report, describes how to isolate and separate human LAPC4 cells from xenografted tumors for further use in downstream protocols. - Protocol 3 describes how to assess expression levels of miR-34a by qRT-PCR in isolated LAPC4 xenograft tumor cells, as seen in Figure 1B of Liu et al., 2011. - [Protocols 4, 5, & 6: Effect of miR-34a on tumor growth and CD44 expression in LAPC4 tumor tissue][14] - Protocol 4 describes how to inject LAPC4 tumor cells infected with a lentivirus containing miR-34a into NOD/SCID mice and measure the resultant tumor size, as seen in Supplemental Figure 5C of Liu et al., 2011. - Protocol 5 This protocol describes Western blot analysis of CD44 levels from tumor tissue derived from Protocol 4, which is similar to figure 4A of the Liu et al., 2011. - Protocol 6 is an additional experiment added by the RP:CB core team and is partially based on Supplemental Figure 4A. It is a quality control check to ensure that increased levels of miR-34a are still present even after the infected LAPC4 cells have been injected into mice to form tumors. - [Protocol 7: Luciferase assays to assess putative miR-34a binding sites in the 3’UTR of CD44][15] - This protocol is the replication of Figure 4D of Liu et al., 2011. It describes how to perform luciferase assays in order to demonstrate that binding of miR-34a to its binding sites in the 3′ UTR of CD44 decreases luciferase activity and tests whether this decrease is abrogated when the seed regions of the putative miR-34a binding sites are mutated. <br> **Meta-analyses:** As a measure of evaluating reproducibility a meta-analysis of each effect was performed. The forest plots, analyses, and data are available [here][16]. Questions about the project can be directed to [contact+rpcb@cos.io][17]. [1]: https://osf.io/e81xl/wiki/home/ [2]: https://elifesciences.org/articles/06434 [3]: http://www.nature.com/articles/nm.2284 [4]: http://www.nature.com/articles/nm.2284 [5]: http://www.nature.com/articles/nm.2284 [6]: http://www.nature.com/articles/nm.2284 [7]: https://elifesciences.org/articles/43511 [8]: https://osf.io/2594d/ [9]: https://www.rstudio.com [10]: https://osf.io/e89mt/ [11]: https://elifesciences.org/articles/06434 [12]: https://osf.io/ts5u6/ [13]: https://osf.io/tkn6c/wiki/home/ [14]: https://osf.io/n9vrz/wiki/home/ [15]: https://osf.io/vzb82/wiki/home/ [16]: https://osf.io/p9xn8/wiki/home/ [17]: mailto:contact+rpcb@cos.io