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. 2014 May 7;9(5):e96239.
doi: 10.1371/journal.pone.0096239. eCollection 2014.

Novel anti-apoptotic microRNAs 582-5p and 363 promote human glioblastoma stem cell survival via direct inhibition of caspase 3, caspase 9, and Bim

Desiree Hunt Floyd  1 , Ying Zhang  1 , Bijan K Dey  1 , Benjamin Kefas  1 , Hannah Breit  1 , Kaitlyn Marks  1 , Anindya Dutta  1 , Christel Herold-Mende  2 , Michael Synowitz  3 , Rainer Glass  4 , Roger Abounader  1 , Benjamin W Purow  1
Affiliations

Novel anti-apoptotic microRNAs 582-5p and 363 promote human glioblastoma stem cell survival via direct inhibition of caspase 3, caspase 9, and Bim

Desiree Hunt Floyd et al. PLoS One. .

Abstract

Glioblastoma is the most common and lethal primary brain tumor. Tumor initiation and recurrence are likely caused by a sub-population of glioblastoma stem cells, which may derive from mutated neural stem and precursor cells. Since CD133 is a stem cell marker for both normal brain and glioblastoma, and to better understand glioblastoma formation and recurrence, we looked for dys-regulated microRNAs in human CD133+ glioblastoma stem cells as opposed to CD133+ neural stem cells isolated from normal human brain. Using FACS sorting of low-passage cell samples followed by microRNA microarray analysis, we found 43 microRNAs that were dys-regulated in common in three separate CD133+ human glioblastomas compared to CD133+ normal neural stem cells. Among these were several microRNAs not previously associated with cancer. We then verified the microRNAs dys-regulated in glioblastoma using quantitative real time PCR and Taqman analysis of the original samples, as well as human GBM stem cell and established cell lines and many human specimens. We show that two candidate oncogenic microRNAs, miR-363 and miR-582-5p, can positively influence glioblastoma survival, as shown by forced expression of the microRNAs and their inhibitors followed by cell number assay, Caspase 3/7 assay, Annexin V apoptosis/fluorescence activated cell sorting, siRNA rescue of microRNA inhibitor treatment, as well as 3'UTR mutagenesis to show luciferase reporter rescue of the most successful targets. miR-582-5p and miR-363 are shown to directly target Caspase 3, Caspase 9, and Bim.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Microarray heatmap of significant miRNA changes.
Relative normalized (log) fold intensity changes for miRNAs in CD133+ GBM sample populations B4, NCH644, and NCH441 are shown in comparison to CD133+ NSCs. Increased miRNAs are shown in red; decreased miRNAs are shown in green.
Figure 2
Figure 2. Real-time PCR verification of miR-582-5p and miR-363 up-regulation in GSCs.
Relative expression of miR-582-5p and miR-363 are depicted for qPCR confirmation of the experimental CD133+ microarray GSC and NPC samples (A and B). Relative expression of miR-582-5p and miR-363 in nine separate human tumor specimens compared to an average of three normal human brain tissue controls with three technical replicates per sample (C and D). Baseline relative expression of miR-582-5p and miR-363 in established (A172 and U373) and stem-like GBM cell lines (0308, 0822, and 1228), as well as normal astrocytes (E and F). An average of two technical replicates and three separate RNA/cDNA samples are shown. Column-to-column p-values are given for A, B, E, and F, taken from one-way analysis of variances followed by Bonferroni’s post-test to compare all columns. Significant differences are indicated thus: *, p<.05, **, p<.01, ****, p<.0001.
Figure 3
Figure 3. Forced expression of anti-miR-363 and anti-miR-582-5p triggers apoptosis.
0822 and U373 GBM stem cell numbers for anti-miR-363 (grey squares), compared to scrambled anti-miRNA negative control (black diamonds) (A). 0822 and 0308 cell numbers for anti-miR-582-5p (grey squares), compared to scrambled anti-miRNA negative control (black diamonds) (C). Immortalized astrocyte and established GBM cell line A172 cell numbers following transfection of miR-582-5p (grey squares) compared to control miRNA (black diamonds), as well as for anti-miR-582-5p (grey squares) compared to anti-miRNA control (black diamonds) for immortalized astrocytes (C). Caspase 3/7 activity and/or Annexin V positivity (shown as % apoptosis) for anti-miR-363 in GBM stem cell lines 0308, 0822 and 1228, compared to scrambled anti-miRNA negative control (D). Caspase 3/7 activity for GBM stem cell lines for anti-miR-582-5p, compared to scrambled anti-miRNA negative control (E). p-values for Student’s t-test analyses are given as *, p<.05, **, p<.01, ***, p<.001. Experiments shown are representative of at least three separate experiments per cell type.
Figure 4
Figure 4. miR-582-5p and miR-363 target Caspases and Bim.
Seed match regions for miR-363 and miR-582-5p in the 3′UTRs of Bim (BCL211), Caspase 3 (CASP3), and Caspase 9 (CASP9), with arrows over mutagenized bases in 3′UTR mutant constructs (A). Immunoblots of Caspase 3 and Caspase 9 for miR-582-5p- or anti-miR-582-5p, three to four days post-transfection of miR-582-5p (+) or control miRNA (–), or anti-miR-582-5p (+) or control scrambled anti-miR (–) Full blots are provided in the supplemental data for single lanes shown, depicting several samples per blot which represent separate transfection wells (B). Immunoblots of Bim and Caspase for miR-363- or anti-miR-363, three to four days post-transfection of miR-582-5p (+) or control miRNA (–), or anti-miR-582-5p (+) or control scrambled anti-miR (–) (C). PARP cleavage immunoblot with forced expression of miR-363 (D). 3′UTR luciferase reporter activitymiR-363 for Caspase 3Bim, or control plasmid (E–F), and for miR-582-5p for Caspase 3 and Caspase 9 or control plasmid (G–H), after correction with control miRNA and control plasmid to yield % maximal activity. 3′-UTR luciferase reporter activity for wild-type and mutagenized BIM and CASP3 target 3′-UTR putative binding sites for miR-582-5p or miR-363 compared to control miR (I). p-values for two-column experiments are derived from Student’s t-tests and for multi-column experiments are derived from two-way ANOVA, with *, p<.05, **, p<.01, ***, p<.001, ****, p<.0001. All blots are representative of several (three or more) separate experiments/transfections, with two to three wells per group per experiment.
Figure 5
Figure 5. Caspase 3 and Bim are critical targets of miR-582-5p and miR-363.
0822 cell numbers Bim siRNA and anti-miR-363 rescue experiment with si- and anti-miRNA scrambled controls (A). Bim immunoblot for similar conditions (B). Caspase 3 immunoblot with siRNA treatment (B) as well as 0822 cell numbers for Caspase 3 siRNA and anti-miR-582-5p rescue experiment with si- and anti-miRNA scrambled controls (C). GBM stem cell numbers following transient transfection with anti-miR-582-5p (grey columns) compared to anti-miRNA scrambled controls, with ZVAD caspase inhibitor treatment rescue (D). One-way ANOVA with Bonferroni’s post-test to compare all columns was used to generate p-values. p-values are represented as *, p<.05, **, p<.01, ***, p<.001 Experiments are representative of three or more separate experiments per panel with up to six separate wells per group.
Figure 6
Figure 6. The apoptotic cascade.
A graphical representation of the miRNAs 582-5p and 363 targeting and decreasing expression of multiple apoptotic pathway components.
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