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, 289 (45), 31121-35

Identification of 14-3-3β Gene as a Novel miR-152 Target Using a Proteome-Based Approach

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Identification of 14-3-3β Gene as a Novel miR-152 Target Using a Proteome-Based Approach

Simon Jasinski-Bergner et al. J Biol Chem.

Abstract

Recent studies demonstrated that miR-152 overexpression down-regulates the nonclassical human leukocyte antigen (HLA) class I molecule HLA-G in human tumors thereby contributing to their immune surveillance. Using two-dimensional gel electrophoresis followed by MALDI-TOF mass spectrometry, the protein expression profile of HLA-G(+), miR-152(low) cells, and their miR-152-overexpressing (miR(high)) counterparts was compared leading to the identification of 24 differentially expressed proteins. These were categorized according to their function and localization demonstrating for most of them an important role in the initiation and progression of tumors. The novel miR-152 target 14-3-3 protein β/α/YWHAB (14-3-3β) is down-regulated upon miR-152 overexpression, although its overexpression was often found in tumors of distinct origin. The miR-152-mediated reduction of the 14-3-3β expression was accompanied by an up-regulation of BAX protein expression resulting in a pro-apoptotic phenotype. In contrast, the reconstitution of 14-3-3β expression in miR-152(high) cells increased the expression of the anti-apoptotic BCL2 gene, enhances the proliferative activity in the presence of the cytostatic drug paclitaxel, and causes resistance to apoptosis induced by this drug. By correlating clinical microarray data with the patients' outcome, a link between 14-3-3β and HLA-G expression was found, which could be associated with poor prognosis and overall survival of patients with tumors. Because miR-152 controls both the expression of 14-3-3β and HLA-G, it exerts a dual role in tumor cells by both altering the immunogenicity and the tumorigenicity.

Keywords: 14-3-3β; Cell Biology; HLA-G; Immunology; MicroRNA (miRNA); Oncogene; Tumor Immunology; miR-152.

Figures

FIGURE 1.
FIGURE 1.
Expression of miR-152, miR-141, and miR-541 in human tumor cell lines. A, qPCR-based detection and quantification of the miR-152 deficiency in JEG-3 cells revealed a 1000-fold reduced expression of miR-152 compared with other human tumor cell lines analyzed (HEK, human embryonic kidney; MZ, renal cell carcinoma cells; melanoma cells). B, qPCR analysis of the miR-152 expression after reconstitution of miR-152 expression in JEG-3 cells. A comparison of the miR-152-expressing transfectants (miR-152high) and the respective mock-control (miR-152low) is shown. The expression of miR-141 (internal control) was not affected. C, miR-152-induced down-regulation of HLA-G in JEG-3 cells after reconstitution of miR-152 expression. As a consequence of the miR-152 reconstitution, the miR-152 target HLA-G was completely down-regulated in the miR-152-expressing transfectants (miR-152high) compared with the mock-control (miR-152low). A representative Western blot of three individual experiments with similar results is shown.
FIGURE 2.
FIGURE 2.
Validation of 14-3-3β as a novel miR-152 target by qPCR and Western blot analysis. A, relative 14-3-3β mRNA expression in the stable miR-152high transfectant of JEG-3 and Buf1088 cells demonstrates a strong down-regulation of the 14-3-3β (YWHAB) transcription. B, protein expression of 14-3-3β in the stable miR-152 transfectants of JEG-3 and Buf1088. Western blot analysis demonstrates a down-regulation of the 14-3-3β protein upon miR-152 overexpression. C, monitoring of 14-3-3β protein in different cell systems. A heterogeneous protein expression of the 14-3-3β protein in the different cell lines was found (HEK, human embryonic kidney; MZ, renal cell carcinoma cells; melanoma cells). A correlation between miR-152 and 14-3-3β could not be observed in the applied cell lines indicating that also other mechanisms could contribute to the regulation of 14-3-3β.
FIGURE 3.
FIGURE 3.
Direct interaction between the 3′-UTR of 14-3-3β and the miR-152. A, luciferase reporter gene assay. The activity of the luc construct lacking any 3′-UTR was not affected by the expression of miR-152 and miR-541 (negative control miR). In contrast, cloning the 14-3-3β 3′-UTR behind the luc reporter gene in combination with the miR-152 transfection leads to a significant reduction of the relative luc reporter gene activity. However, co-transfection of miR-541 as nonsense control and of the pmR-mock vector had no effect on the relative luc reporter gene activity. The deletion (Δ) of the in silico predicted miR-152-binding site completely restored the reductive effect of miR-152 on the 14-3-3β 3′-UTR (*, t test; p < 0.05). B, to block miR-152 activity, an miR-152-specific decoy was cloned according to Haraguchi et al. (25) and combined in the luc reporter gene assay, resulting in an increased luc activity of the 14-3-3β 3′-UTR upon miR-152 blocking compared with respective mock controls. C, applying a novel miR enrichment assay (26), the direct interaction of miR-152 and the 14-3-3β 3′-UTR can be demonstrated. The input shows the available amount of miR-152 and as internal control miR-141 in the used cell lysate. There was no enrichment of miR-152 in the absence of the 3′-UTR as bait (<10 copies = background). In contrast with the HLA-G 3′-UTR (positive control) and even more effective with the 14-3-3β 3′-UTR as bait, miR-152 was strongly enriched out of the cell lysate. The internal negative control miR-141, present in the input, was not enriched. D, scheme of the in silico analysis for putative miR-152-binding site within the 14-3-3β (YWHAB) 3′-UTR.
FIGURE 4.
FIGURE 4.
Reconstitution of the 14-3-3β expression and apoptosis assays. A and B, analysis of the reconstitution of the 14-3-3β expression in the miR-152 transfectants of JEG-3 by stable transfection with an expression vector (pExp) encoding the 14-3-3β coding sequence without 3′-UTR by qPCR (A) and Western blot (B). C, miR-152-induced up-regulation of BAX protein. Before the detection of BAX protein, the transfectants were cultured for 72 h in the absence and presence of 25 nm paclitaxel. Increased levels of the proapoptotic protein BAX were detected in all miR-152high transfectants in the absence of paclitaxel but with no concomitant effect on the cleaved caspase 3 levels. In the presence of paclitaxel, reconstitution of the anti-apoptotic 14-3-3β protein reduced the BAX protein level but had no effect on cleaved caspase 3. D, decreased sensitivity toward drug treatment with paclitaxel upon 14-3-3β reconstitution. Cells were grown for 72 h in the absence or presence of various concentrations of paclitaxel (0–20 μm) and analyzed via XTT assay using the XTT cell proliferation kit (Roche Applied Science) according to the manufacturer's protocol. E, detection of altered apoptosis sensitivity in the distinct JEG-3 transfectants using annexin V and 7-amino-actinomycin D staining. Before the detection of the apoptotis rates, the transfectants were cultured for 72 h in the absence and presence of 25 nm paclitaxel. Whereas the apoptosis sensitivity of untreated transfectants (DMSO control) remained unaffected, a reduction of the paclitaxel-induced apoptosis was detected after 14-3-3β reconstitution. *, t test; p < 0.05. F, up-regulation of BCL2 gene expression after 14-3-3β reconstitution was detected after 72 h of incubation in the absence and presence of 25 nm paclitaxel. *, t test; p < 0.05.
FIGURE 5.
FIGURE 5.
Role of 14-3-3β for cell proliferation. A, flow cytometry analysis of CFSE-stained JEG-3 transfectants after 72 h of incubation in the absence or presence of 25 nm paclitaxel revealed a paclitaxel-induced reduction of the proliferation rate of the miR-152-expressing transfectants due to 14-3-3β down-regulation and the reversal of this effect due to 14-3-3β reconstitution, whereas the proliferation rate of untreated transfectants (DMSO control) remained unaffected. The number provided within the given profile highlights the percentage of proliferating cells. The bar chart at right summarizes the results based on three independent experiments. t test; p < 0.05. B, analyzing the cell cycle revealed that the reconstitution of the 14-3-3β protein in the miR-152 overexpressing transfectants enhances the percentage of mitotic cells (M-phase) t test; p < 0.05. C, quantitation of the gene expression of distinct cyclins and cyclin-dependent kinases involved in the cell cycle regulation showed a strong increase in the expression of CCNE and CDK2 resulting in an enhanced transition from G1 to S phase. *, t test; p < 0.05; **, t test; p < 0.005.
FIGURE 6.
FIGURE 6.
Correlation of the expression of HLA-G and 14-3-3β with overall patient survival in distinct tumor entities. A–H, higher the expression of the miR-152 targets HLA-G and 14-3-3β, the worse is the overall survival of the patients. A and B, neuroblastoma; C and D, glioma; E and F, lung cancer; G and H, osteosarcoma. The plots are based on transcriptional quantifications. The analysis was performed using the on-line database R2, microarray analysis and visualization platform.
FIGURE 7.
FIGURE 7.
Annotation clustering of the differentially expressed proteins upon miR-152 overexpression in JEG-3 cells identified by 2DE-based proteomic profiling and mass spectrometry. The differentially expressed proteins upon miR-152 overexpression in JEG-3 cells identified by 2DE-based proteomic profiling and mass spectrometry were grouped according to their GO annotations for their biologic process (A), cellular localization (B), and to their molecular functions (C).
FIGURE 8.
FIGURE 8.
Principle of the miR enrichment assay miTRAP (A), hypothesis of the dual role of miR-152 as tumor suppressor (B), and influence of 14-3-3β on the cell cycle (C). A, scheme of the miR enrichment assay miTRAP. An in vitro transcribed synthetic RNA construct, consisting of the 3′-UTR of interest and four MS2 loops, is employed as bait for specific miR enrichment out of a cell lysate (MZ2905RC; HLA-G mRNA+/protein−). These RNA constructs are loaded on amylose beads for a specific enrichment of miRs by applying a fusion protein of MS2 loop-binding protein and maltose-binding protein (26). B, working hypothesis demonstrating that a defect in the expression of the miR-148 family affects the expression of HLA-G and 14-3-3β altering the immunogenicity and the tumorigenicity for tumor cells. C, scheme of the cell cycle and involved cyclins and cyclin-dependent kinases; altered after Bonelli et al. (52).

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