MicroRNA-21 correlates with tumorigenesis in malignant peripheral nerve sheath tumor (MPNST) via programmed cell death protein 4 (PDCD4)

Abstract

Purpose: We investigated the miRNA profile in peripheral nerve tumors and clarified the involvement of miRNA in the development and progression of MPNST in comparison with neurofibroma (NF). In addition, we attempted to seek associations between the miRNA and their potential targets in MPNST.

Methods: Global miRNA expression profiling was investigated for clinical samples of 6 MPNSTs and 6 NFs. As detected by profiling analysis, the expressions of miR-21 in clinical samples of 12 MPNSTs, 11 NFs, and 5 normal nerves, and 3 MPNST cell lines were compared using quantitative real-time reverse transcription PCR. MPNST cell line (YST-1) was transfected with miR-21 inhibitor to study its effects on cell proliferation, caspase activity, and the expression of miR-21 targets.

Results: Analysis of miRNA expression profiles in MPNST and NF revealed significantly altered expression levels of nine miRNAs, one of those, miR-21, and its putative target, programmed cell death protein 4 (PDCD4), were selected for further studies. miR-21 expression level in MPNST was significantly higher than that in NF (P < 0.05). In MPNST cells, transfection of miR-21 inhibitor significantly increased caspase activity (P < 0.01), significantly suppressed cell growth (P < 0.05), and upregulated protein level of PDCD4, indicating that miR-21 inhibitor could induce cell apoptosis of MPNST cells.

Conclusions: These results suggest that miR-21 plays an important role in MPNST tumorigenesis and progression through its target, PDCD4. MiR-21 and PDCD4 may be candidate novel therapeutic targets against the development or progression of MPNSTs.

Fig. 1

Hierarchical clustering. miRNA expression was examined in 6 MPNSTs and 6 NFs. Twelve samples were clustered according to the expression profile of 9 miRNAs that were differentially expressed between MPNSTs and NFs. P values <0.01 were considered statistically significant. The colored bar at the top denotes the relative expression values seen in the cluster table

Fig. 2

Verification of 5 miRNAs expression using quantitative RT-PCR in clinical samples from MPNSTs, NFs, and normal nerves; a miR-21, b miR-125b, c miR-127, d miR-135b, e miR-302d. MPNST cell lines. The expression level of miR-21 in clinical samples from MPNSTs was significantly higher that than in NFs. miR-21 expression in MPNST cell lines was also significantly higher than that in normal nerves (f). *P < 0.05, **P < 0.01

Fig. 3

Monitoring transfection efficiency in YST-1 cells. Transfection efficiencies were calculated by the ratio of FAM-labeled cells via a phase-contrast and b fluorescence imaging

Fig. 4

Influence of miR-21 inhibitor in YST-1 cells. a miR-21 expression level in transfected cells. The expression level of miR-21 was significantly downregulated 24 h after transfection with miR-21 inhibitor in YST-1 cells. b Apoptotic activity using caspase 3/7 assay 48 h after transfection. Transfection with anti-miR-21 significantly increased apoptosis. c Cell proliferation assay using CellTilter-Glo 72 h after transfection. Cells transfected with miR-21 inhibitor showed significantly lower proliferation. Inhibitor miR-21 inhibitor, NC negative control. *P < 0.05, **P < 0.01

Fig. 5

PDCD4 protein expression in YST-1 cells. Western blotting detected that transfection with miR-21 inhibitor upregulated PDCD4 expression. The membranes were blotted with PDCD4 and GAPDH antibody. Intensities of each protein band were detected using ECL Detection System and analyzed using Image J software (NIH). The results are presented as fold change compared to their respective untreated controls (mock). Inhibitor miR-21 inhibitor, NC negative control

Fig. 6

PDCD4 protein expression in clinical samples of MPNSTs, NFs and normal nerves. The expression of PDCD4 protein in NFs and normal nerves was significantly higher than that in MPNSTs. *P < 0.05, **P < 0.01