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. 2014 Oct 9;9(10):e109124.
doi: 10.1371/journal.pone.0109124. eCollection 2014.

MiR-181b-5p downregulates NOVA1 to suppress proliferation, migration and invasion and promote apoptosis in astrocytoma

Feng Zhi  1 , Qiang Wang  2 , Danni Deng  1 , Naiyuan Shao  2 , Rong Wang  1 , Lian Xue  1 , Suinuan Wang  2 , Xiwei Xia  2 , Yilin Yang  1
Affiliations

MiR-181b-5p downregulates NOVA1 to suppress proliferation, migration and invasion and promote apoptosis in astrocytoma

Feng Zhi et al. PLoS One. .

Erratum in

Abstract

MicroRNAs (miRNAs) are small, short noncoding RNAs that modulate the expression of numerous genes by targeting their mRNA. Numerous abnormal miRNA expression patterns are observed in various human malignancies, and certain miRNAs can act as oncogenes or tumor suppressors. Astrocytoma, the most common neuroepithelial cancer, represents the majority of malignant brain tumors in humans. In our previous studies, we found that the downregulation of miR-181b-5p in astrocytomas is associated with a poor prognosis. The aim of the present study was to investigate the functional role of miR-181b-5p and its possible target genes. miR-181b-5p was significantly downregulated in astrocytoma specimens, and the reduced expression of miR-181b-5p was inversely correlated with the clinical stage. The ectopic expression of miR-181b-5p inhibited proliferation, migration and invasion and induced apoptosis in astrocytoma cancer cells in vitro. The NOVA1 (neuro-oncological ventral antigen 1) gene was further identified as a novel direct target of miR-181b-5p. Specifically, miR-181b-5p bound directly to the 3'-untranslated region (UTR) of NOVA1 and suppressed its expression. In clinical specimens, NOVA1 was overexpressed, and its protein levels were inversely correlated with miR-181b-5p expression. Furthermore, the changing level of NOVA1 was significantly associated with a poor survival outcome. Similar to restoring miR-181b-5p expression, downregulating NOVA1 inhibited cell growth, migration and invasion. Overexpression of NOVA1 reversed the inhibitory effects of miR-181b-5p. Our results indicate that miR-181b-5p is a tumor suppressor in astrocytoma that inhibits tumor progression by targeting NOVA1. These findings suggest that miR-181b-5p may serve as a novel therapeutic target for astrocytoma.

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Conflict of interest statement

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

Figures

Figure 1
Figure 1. miR-181b-5p is downregulated in astrocytoma tissue samples.
The miR-181b-5p expression was normalized to U6. (A) Relative expression of miR-181b-5p in NAT and astrocytoma tissues. (B) Relative expression of miR-181b-5p in different WHO grades of cancer tissues.
Figure 2
Figure 2. The role of miR-181b-5p in cell proliferation, migration, invasion and apoptosis in vitro.
(A) Overexpression or knockdown of miR-181b-5p. pre-ncRNA, pre-miR-181b-5p, anti-ncRNA or anti-miR-181b-5p was transfected. The intercellular levels of miR-181b-5p were evaluated by qRT-PCR at 24 h after transfection. For comparison, the expression levels of miR-181b-5p in pre-ncRNA- or anti-ncRNA transfected cells were arbitrarily set at 1. The results are presented as the mean ± SD of three independent experiments (*** p<0.001). (B) The role of miR-181b-5p in cell proliferation. An MTT cell viability assay was performed at 0, 24, 48 and 72 h after the transfection of U251 cells with equal concentrations of pre-ncRNA, pre-miR-181b-5p, anti-ncRNA and anti-miR-181b-5p. For comparison, the expression levels of miR-181b-5p in pre-miR-181b-5p- or anti-miR-181b-5p transfected cells were compared with their respective negative controls (* p<0.05, *** p<0.001). The experiment was repeated three times. (C) Wound-healing assays of U251 cells treated with equal concentrations of pre-ncRNA, pre-miR-181b-5p, anti-ncRNA and anti-miR-181b-5p. The wound gaps were photographed and measured. The images shown are representative images from three independent experiments. (D) Transwell assays of U251 cells treated with equal concentrations of pre-ncRNA, pre-miR-181b-5p, anti-ncRNA and anti-miR-181b-5p. The images shown are representative images from three independent experiments, and a statistical analysis was performed (mean ± SD, *** p<0.001). (E) The role of miR-181b-5p in apoptosis. U251 cells were transfected with equal concentrations of pre-ncRNA, pre-miR-181b-5p, anti-ncRNA and anti-miR-181b-5p. The experiment was repeated three times, and representative data are shown.
Figure 3
Figure 3. NOVA1 is a direct target gene of miR-181b-5p.
(A) A schematic description of the hypothesized duplexes formed by interactions between the NOVA1 3′-UTR binding sites and miR-181b-5p. The predicted free energy of each hybrid is indicated. (B) Direct recognition of the NOVA1 3′-UTR by miR-181b-5p. Firefly luciferase reporters containing either wt or mut NOVA1 3′-UTRs were co-transfected into U251 cells with pre-miR-181b-5p, anti-miR-181b-5p and their corresponding negative controls. The parental luciferase plasmid was also transfected as a control. At 24 h post-transfection, the cells were assayed using luciferase assay kits. The results are presented as the mean ± SD of three independent experiments (*** p<0.001). (C) Quantitative real time-PCR analysis of NOVA1 mRNA expression levels in U251 cells treated with pre-ncRNA, pre-miR-181b-5p, anti-ncRNA and anti-miR-181b-5p. The results shown represent data from three independent experiments (*** p<0.001). (D) Representative western blots showing NOVA1 protein levels in U251 cells treated with pre-ncRNA, pre-miR-181b-5p, anti-ncRNA and anti-miR-181b-5p. (E) Representative western blots showing NOVA1 protein levels in NAT samples and WHO I-IV astrocytomas. (F) Statistical analysis of relative expression of NOVA1 in NAT samples and WHO I-IV astrocytomas. The expression of NOVA1 in NAT samples were arbitrarily set at 1. (G) Spearman's correlation analysis was used to determine the correlation between the expression levels of NOVA1 and miR-181b-5p in human astrocytoma specimens. (H) The relationship between NOVA1 expression and astrocytoma patient survival time.
Figure 4
Figure 4. NOVA1 downregulation inhibited cell proliferation, migration and invasion and promoted apoptosis in vitro.
(A) Downregulation of NOVA1 decreased cell growth (* p<0.05, ** p<0.01). The experiment was repeated three times. (B) Downregulation of NOVA1 decreased cell migration ability. (C) Downregulation of NOVA1 decreased cell invasion ability. The images shown are representative images from three independent experiments, and a statistical analysis was performed (mean ± SD, *** p<0.001). (D) Downregulation of NOVA1 promoted apoptosis.
Figure 5
Figure 5. Overexpression of NOVA1 reversed the inhibitory effects of miR-181b-5p.
(A) Cell proliferation assay. An MTT cell viability assay was performed at 0, 24, 48 and 72 h after the transfection of U251 cells with control vector + pre-ncRNA, control vector + pre-miR-181b-5p, NOVA1 vector + pre-ncRNA, and NOVA1 vector + pre-miR-181b-5p. The experiment was repeated three times. (B) Cell apoptosis assay. U251 cells were transfected with control vector + pre-ncRNA, control vector + pre-miR-181b-5p, NOVA1 vector + pre-ncRNA, and NOVA1 vector + pre-miR-181b-5p. The experiment was repeated three times, and representative data were shown.
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This work was supported by National Natural Science Foundation of China (31071046, 81302197), Changzhou Social Development Project (CS20092015, CS20102010), Changzhou Health Bureau Project (ZD200903, ZD201007), Changzhou Science Technology Bureau Guiding Project (CY20119004). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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