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. 2015 Jul 20:8:1773-83.
doi: 10.2147/OTT.S72457. eCollection 2015.

miR-32 functions as a tumor suppressor and directly targets SOX9 in human non-small cell lung cancer

Dan Zhu  1 , Hui Chen  1 , Xiguang Yang  1 , Weisong Chen  1 , Linying Wang  1 , Jilin Xu  1 , Long Yu  1
Affiliations

miR-32 functions as a tumor suppressor and directly targets SOX9 in human non-small cell lung cancer

Dan Zhu et al. Onco Targets Ther. .

Retraction in

Abstract

Purpose: MicroRNA-32 (miR-32) is dysregulated in certain human malignancies and correlates with tumor progression. However, its expression and function in non-small cell lung cancer (NSCLC) remain unclear. Thus, the aim of this study was to explore the effects of miR-32 expression on NSCLC tumorigenesis and development.

Methods: Using real-time quantitative reverse-transcription polymerase chain reaction (qRT-PCR), we detected miR-32 expression in NSCLC cell lines and primary tumor tissues. The association of miR-32 expression with clinicopathological factors and prognosis was also analyzed. Then, the effects of miR-32 expression on the biological behavior of NSCLC cells were investigated. Finally, the potential regulatory effect of miR-32 on SOX9 expression was confirmed.

Results: miR-32 expression levels were significantly downregulated in NSCLC compared with the corresponding noncancerous lung tissues (P<0.001). In addition, decreased miR-32 expression was significantly associated with lymph node metastasis (P=0.002), advanced tumor/nodes/metastasis (TNM) classification stages (P<0.001), and shorter overall survival (P<0.001). Multivariate regression analysis corroborated that downregulated miR-32 expression was an independent unfavorable prognostic factor for NSCLC patients. In vitro studies demonstrated that miR-32 overexpression reduced A549 cell proliferation, migration, and invasion, and promoted apoptosis. Furthermore, SOX9 was confirmed as a direct target of miR-32, using a luciferase reporter assay.

Conclusion: These findings indicate that miR-32 may act as a tumor suppressor in NSCLC and could serve as a novel therapeutic agent for miR-based therapy.

Keywords: apoptosis; invasion; migration; prognosis; proliferation.

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Figures

Figure 1
Figure 1
Expression of miR-32 and SOX9 in NSCLC tissues and cell lines. Notes: (A) miR-32 expression was significantly lower in NSCLC tissues than in the corresponding noncancerous tissues. miR-32 expression levels were calculated by the 2−∆Ct method and normalized to U6 small nuclear RNA. (B) miR-32 expression was downregulated in the NSCLC cell lines A549, H460, 95D, and HCC827 compared with NLECs. (C) Relative SOX9 protein levels in NSCLC and corresponding non cancerous tissues. SOX9 protein levels were measured by Western blot analysis and normalized to β-actin. (D) SOX9 protein levels in NSCLC cells were higher than in NLECs. (E) The inverse correlation of SOX9 protein levels with miR-32 expression was examined by Pearson correlation analysis. *P<0.05; **P<0.01. Abbreviations: miR-32, microRNA-32; NLECs, normal lung epithelial cells; NSCLC, non-small cell lung cancer.
Figure 2
Figure 2
Overall survival curves for two groups defined by low and high expression of miR-32 in patients with non-small cell lung cancer. Notes: Low miR-32 expression levels were significantly associated with poor outcomes (P<0.001, log-rank test). Abbreviation: miR-32, microRNA-32.
Figure 3
Figure 3
Effects of miR-32 mimic or inhibitor transfection on the biological behaviors of A549 and 95D cells. Notes: (A) qRT-PCR analysis confirmed increased miR-32 expression in A549 cells transfected with miR-32 mimics and decreased miR-32 expression in 95D cells transfected with miR-32 inhibitors. U6 RNA was used as an internal control. **P<0.01; ***P<0.001. (B) An MTT assay showed that miR-32 expression reduced cell proliferation in vitro. The data represent the mean ± SD of the experiments performed in triplicate. **P<0.01. (C) Cell apoptosis was detected by flow cytometric analysis after transfection with miR-32 mimics, miR-32 inhibitors, or a negative control. (D) A Transwell® invasion assay demonstrated that the up-regulation of miR-32 expression impeded the invasion of A549 cells, whereas the transfection of 95D cells with miR-32 inhibitors promoted cell invasion. (E) A scratch migration assay confirmed the inhibitory effect of miR-32 on NSCLC cell migration. **P<0.01. Abbreviations: miR-32, microRNA-32; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium; NSCLC, non-small cell lung cancer; qRT-PCR, quantitative reverse-transcription polymerase chain reaction; SD, standard deviation; OD, optical density; NC, negative control.
Figure 3
Figure 3
Effects of miR-32 mimic or inhibitor transfection on the biological behaviors of A549 and 95D cells. Notes: (A) qRT-PCR analysis confirmed increased miR-32 expression in A549 cells transfected with miR-32 mimics and decreased miR-32 expression in 95D cells transfected with miR-32 inhibitors. U6 RNA was used as an internal control. **P<0.01; ***P<0.001. (B) An MTT assay showed that miR-32 expression reduced cell proliferation in vitro. The data represent the mean ± SD of the experiments performed in triplicate. **P<0.01. (C) Cell apoptosis was detected by flow cytometric analysis after transfection with miR-32 mimics, miR-32 inhibitors, or a negative control. (D) A Transwell® invasion assay demonstrated that the up-regulation of miR-32 expression impeded the invasion of A549 cells, whereas the transfection of 95D cells with miR-32 inhibitors promoted cell invasion. (E) A scratch migration assay confirmed the inhibitory effect of miR-32 on NSCLC cell migration. **P<0.01. Abbreviations: miR-32, microRNA-32; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium; NSCLC, non-small cell lung cancer; qRT-PCR, quantitative reverse-transcription polymerase chain reaction; SD, standard deviation; OD, optical density; NC, negative control.
Figure 4
Figure 4
SOX9 is a direct target of miR-32. Notes: (A) miR-32-binding sites in the SOX9 3′ UTR region. SOX9-mut indicates the SOX9 3′ UTR with mutations in miR-32-binding sites. (B) Western blotting showed that the transfection of miR-32 decreased SOX9 protein expression. (C) Relative luciferase assay comparing the pGL3-SOX9 and pGL3-SOX9-mut vectors in A549 cells. Firefly luciferase activity was normalized to Renilla luciferase activity. *P<0.05. Abbreviations: miR-32, microRNA-32; UTR, untranslated region; WT, wild-type.
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