MiR-133b Targets Antiapoptotic Genes and Enhances Death Receptor-Induced Apoptosis

Despite the importance of microRNAs (miRs) for regulation of the delicate balance between cell proliferation and death, evidence for their specific involvement during death receptor (DR)-mediated apoptosis is scarce. Transfection with miR-133b rendered resistant HeLa cells sensitive to tumor necrosis factor-alpha (TNFα)-induced cell death. Similarly, miR-133b caused exacerbated proapoptotic responses to TNF-related apoptosis-inducing ligand (TRAIL) or an activating antibody to Fas/CD95. Comprehensive analysis, encompassing global RNA or protein expression profiling performed by microarray experiments and pulsed stable isotope labeling with amino acids in cell culture (pSILAC), led to the discovery of the antiapoptotic protein Fas apoptosis inhibitory molecule (FAIM) as immediate miR-133b target. Moreover, miR-133b impaired the expression of the detoxifying protein glutathione-S-transferase pi (GSTP1). Expression of miR-133b in tumor specimens of prostate cancer patients was significantly downregulated in 75% of the cases, when compared with matched healthy tissue. Furthermore, introduction of synthetic miR-133b into an ex-vivo model of prostate cancer resulted in impaired proliferation and cellular metabolic activity. PC3 cells were also sensitized to apoptotic stimuli after transfection with miR-133b similar to HeLa cells. These data reveal the ability of a single miR to influence major apoptosis pathways, suggesting an essential role for this molecule during cellular transformation, tumorigenesis and tissue homeostasis

Transfection of HeLa cells with miR-133b results in enhanced caspase-dependent loss of membrane integrity after DR ligation.

<p>Cells were transfected with miR-133b alone or together with ctrl miR. After 48 h, cells were left untreated (Unstim) or stimulated for 4 h with either 20 ng/ml TNFα, 100 ng/ml αFas/CD95 or 20 ng/ml rhTRAIL. In order to assess caspase dependency, 0.01% DMSO (vehicle) (left histogram column) or Z-VAD-FMK (50 µM) (right histogram column) were added to the cells. Following stimulation, both adherent and suspension cells were collected, stained with propidium iodide (PI) and analyzed by flow cytometry. Samples were compared to equally treated ctrl miR-transfected cells (black histogram). Results are representative of at least three independent experiments.</p

Genes downregulated after transfection of HeLa cells with miR-133b and TNFα stimulation and predicted to be potential miR targets.¹

1<p>According to the online database miRecords and predicted at least by three different algorithms.</p

Samples from human prostate cancer patients have decreased expression of miR-133b.

<p>Scatter dot-plot of miR-133b expression. RT-qPCR was used to determine miR-133b expression in 69 primary prostate cancer tissues and corresponding normal adjacent tissue. Lines represent mean expression values. MiR-133b was normalized to miR-130b, since it shows a very stable expression pattern both in tumors and normal tissue.</p

Prostate cancer cells treated with miR-133b show impaired vitality.

<p>(A) MTT proliferation assay. PC3 cells were transfected with ctrl miR (•) or, miR-133b together with ctrl αmiR (▪) or with a specific αmiR-133b (▴). Cellular viability and proliferation were determined 0, 48, 96 and 144 h post-transfection as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035345#s4" target="_blank">Material and Methods</a>. (B) 3′-UTR targeting reporter assay. PC3 cells were transfected with miR mimics and a luciferase reporter plasmid encompassing the complete 3′-UTR of FAIM (psiCHECK2-FAIM) or the 3′-UTR missing the miR-133b binding site (psiCHECK2-FAIM-mut). The empty psiCHECK2 vector was used as a negative control. Error bars indicate standard deviation. (C) Congruent to (B) except that luciferase assays were performed with a construct harboring the GSTP1 3′-UTR.</p

miR-133b acts proapoptotically on HeLa cells stimulated with death receptor (DR) ligands.

<p>Cells were transfected with miR-133b alone or together with a control antimiR (ctrl αmiR) or a specific miR-133b inhibitor (αmiR-133b). After 48 h, cells were either left untreated (Unstim) or stimulated for 4 h with 20 ng/ml tumor necrosis factor-alpha (TNFα), 100 ng/ml of a cross-linking activating antiFas antibody (αFas/CD95) or 20 ng/ml recombinant human TRAIL (rhTRAIL). (A) Treated cells were harvested, stained and scanned by flow cytometry for the presence of cleaved active caspase 8 (upper graph) and 3 (lower graph). 7-Amino-actinomycin D (7-AAD) served for exclusion of cells with compromised membrane integrity from the caspase activation quantification assay. Cells transfected with ctrl miR alone were used as reference. (B) Western blot analysis of poly [ADP ribose] polymerase (PARP-1) in transfected, unstimulated cells (upper panel) and TNFα-, αFas/CD95- or rhTRAIL-treated cells (lower panel). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal loading control. Graphs are representative of at least three independent experiments. Asterisk represents p<0.01. Errors bars indicate standard deviation.</p

miR-133b induces increased apoptosis in DR receptor stimulated PC3 cells.

<p>Cells were transfected with miR-133b (red) or a control miR (ctrl miR, black), as reference. After 48 h, cells were either left untreated (Unstim) or stimulated for 4 h with 20 ng/ml tumor necrosis factor-alpha (TNFα), 100 ng/ml of a cross-linking activating antiFas antibody (αFas/CD95) or 20 ng/ml recombinant human TRAIL (rhTRAIL). Treated cells were harvested, stained and scanned by flow cytometry for the presence of cleaved active caspase 8 (left) and caspase 3 (right). 7-Amino-actinomycin D (7-AAD) was used for exclusion of cells with compromised membrane integrity from the caspase activation quantification assay.</p

miR-133b controls expression of glutathione-<i>S</i>-transferase pi (GSTP1).

<p>(A) miR-133b target sites within the 3′-UTR of GSTP1 as predicted by TargetScan and MicroCosm Targets. (B) 3′-UTR targeting assay. HeLa cells were cotransfected with different combinations of miR mimics and a luciferase reporter plasmid harboring the complete 3′-UTR of GSTP1 (psiCHECK2-GSTP1) or the 3′-UTR missing the miR-133b binding site (psiCHECK2-GSTP1-mut). Template plasmid (psiCHECK2) was used as a negative and normalization control. Activity of the 3′-UTR-dependent luciferase (<i>Renilla reniformis</i>) was measured 48 h post-transfection and normalized for transfection efficiency to the one produced by the miR- and 3′-UTR-independent luciferase (<i>Photinus pyralis</i>). Error bars indicate standard deviation. (C) Western blot and densitometric analysis of GSTP1 expression. Cells were transfected with miR-133b alone or together with ctrl αmiR or a specific αmiR-133b. After 48 h, cellular protein lysates were prepared and GSTP1 expression was assessed by Western blot. GAPDH was used as an internal loading standard. Ctrl miR-transfected cells were used as a reference for densitometric quantification of protein band intensity. (D) qPCR analysis. Cells were transfected for 48 h before total RNA was isolated, reverse transcribed and analyzed by qPCR for the expression of GSTP1. HuPO was used as the housekeeping gene for internal normalization. Fold-change (FCH) values are shown relative to mock-transfected cells incubated under the same conditions. All graphs are representative of at least three independent experiments.</p

Apoptosis regulatory proteins downregulated in miR-133b-transfected TNFα-stimulated HeLa cells.

1<p>Compared to ctrl miR-transfected and TNFα-stimulated cells. P-value<0.01.</p>2<p>As predicted by either TargetScan or MicroCosm Targets.</p