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. 2017 Jul 13;18(9):1604–1617. doi: 10.15252/embr.201643735

Figure 5. The miR‐15 family targets cyclins E1 and D3 in pre‐B cells.

Figure 5

  • A
    Schematic illustration of the applied strategy for miR‐15 target identification. Genes upregulated (more than 1.25‐fold, P < 0.05) in the microarray data of miR‐15 sponge versus scrambled sponge samples, both in the presence and in the absence of IL‐7, were filtered for predicted, conserved miR‐15a, miR‐15b, and miR‐16 binding sites in their 3′‐UTR using the PicTar and miRanda algorithms.
  • B
    Quantitative PCR for cyclin E1 and cyclin D3 gene expression in control and in miR‐15 sponge pre‐B cells, respectively, in the presence of IL‐7 and upon IL‐7 withdrawal (36 h). Individual experiments were normalized to the scrambled sponge; +IL‐7 sample. Groups were compared by a paired t‐test; ***P < 0.001. Bars represent means of 10 independent experiments ± SD.
  • C, D
    Direct regulation of cyclin E1, but indirect regulation of cyclin D3, by the miR‐15 family. Pre‐B cells selected for expression of a control construct encoding GFP without a 3′‐UTR, a positive control with two perfect miR‐16 target sites (TS) as well as reporters with the 3′‐UTRs of cyclin E1 and cyclin D3 were transduced with an empty vector as well as with expression constructs for miR‐15a_16‐1 and miR‐15b_16‐2 clusters (C). In a reverse approach, the reporter cells were furthermore transduced with the scrambled sponge as a control or with the sponge construct targeting the miR‐15 family (D). Individual bars display the ratio in GFP MFI comparing transduced and non‐transduced cells measured by flow cytometry after 48 h (ratios were normalized to the empty vector control, respectively, which was set as 1). Values (combined for miR‐15a_16‐1 and miR‐15b_16‐2 in C) were compared by a one‐way ANOVA followed by Bonferroni testing; ***P < 0.001. Bars represent means ± SD of least three independent experiments.
  • E
    Pre‐B cells selected for the scrambled sponge, the miR‐15 sponge, or the empty control vector were cultured with IL‐7 or without IL‐7 for 36 h before lysis, SDS–PAGE, and Western blotting with anti‐cyclin D3, anti‐cyclin E1, and anti‐actin antibodies. Shown data are representative for three independent experiments.

Source data are available online for this figure.