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. 2015 Aug 5;34(1):76.
doi: 10.1186/s13046-015-0192-z.

microRNA-93 promotes cell proliferation via targeting of PTEN in Osteosarcoma cells

Masanori Kawano  1 , Kazuhiro Tanaka  2 , Ichiro Itonaga  3 , Shinichi Ikeda  4 , Tatsuya Iwasaki  5 , Hiroshi Tsumura  6
Affiliations

microRNA-93 promotes cell proliferation via targeting of PTEN in Osteosarcoma cells

Masanori Kawano et al. J Exp Clin Cancer Res. .

Abstract

Background: Aberrant microRNA (miRNA) expression plays an essential role in osteosarcoma (OS) pathogenesis. Recent studies have shown that dysregulation of miRNA expression is associated with increased tumorigenesis and poor prognosis in several types of cancers, including OS. The aim of this study was to investigate the relevant microRNAs involved in the development of OS.

Methods: To explore possible oncogenic factors in OS, we used a microarray-based approach to profile changes in the expression of miRNAs and their target mRNAs in five OS cell lines and human mesenchymal stem cells (hMSCs). An miRNA, miR-93, was significantly up-regulated, whereas phosphatase and tensin homologue (PTEN) expression was significantly down-regulated in all tested OS cells, when compared with hMSCs.

Results: When anti-miR-93 was transfected into OS cell lines, PTEN expression was greatly increased, suggesting that PTEN might be a target of miR-93 in ES cells. The expression of phosphorylated Akt protein, which is known to be inversely correlated with that of PTEN, was significantly down-regulated in anti-miR-93-transfected cells. Furthermore, transfection of anti-miR-93 inhibited the proliferation and cell cycle progression of ES cells. In addition, the down-regulation of miR-93 in these cells significantly suppressed tumor growth in vivo.

Conclusion: Ectopic expression of miR-93 decreased PTEN protein levels. Furthermore, miR-93 increased proliferation and decreased apoptosis in OS cells, whereas its silencing in these cells inhibited such carcinogenic processes. Taking these observations together, miR-93 can be seen to play a critical role in carcinogenesis through suppression of PTEN, and may serve as a therapeutic target for the treatment of OS.

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Figures

Fig. 1
Fig. 1
microRNA and cDNA array analysis in OS cell lines. a Heat map of genome-wide miRNA profile in five OS cell lines and hMSCs. miR-93 was down-regulated in all five OS cell lines. b Whole genome mRNA profile and extraction of PTEN and related genes as same pathways in OS cells and hMSCs. The color bar represented the grades of the relative expression levels; increase was represented by red while decrease was indicated by blue
Fig. 2
Fig. 2
miR-93 inhibits PTEN mRNA expression in Saos cells and silencing of PTEN with anti-miR-93 in OS cells. a Predicted binding sites of miR-93 at the 3′UTR site of PTEN, as aligned by Target Scan and BLAST. b Scheme and sequence of the intact miR- 93 (Wt) and its mutant (Mut). (c) After actinomycinD treatment, the miRNA expression level of miR-93 in the negative control-miR, miR-93 and miR-93 mutant was measured by qRT-PCR. d After actinomycinD treatment, the mRNA expression level of PTEN after transfection of negative control-miR, miR-93 and miR-93 mutant was measured by qRT-PCR. e Up-regulation of PTEN using anti- miR-93 and PTEN-expression vector in ES cells. f Densitometry quantification of PTEN protein after transfection of anti-miR-93. g Transfection of PTEN expression vector in ES cells increases the expression of PTEN protein. h The quantification of PTEN protein after transfection of PTEN expression vector. ANOVA was carried out to statistically analyze the results. (**) P < 0.01
Fig. 3
Fig. 3
Cell proliferation assay performed to evaluate the anti-proliferation effect. Cell proliferation assay to evaluate the antiproliferation effect of anti-miR-93 (a) and (b) and PTEN vector (c and d) in OS cells. Error bars represent mean ± S.D. from three independent experiments. The two-tail student t-test was employed to statistically analyze the results: (**) P < 0.01
Fig. 4
Fig. 4
A western blot analysis of PTEN, Akt and other factors related to cell cycle and apoptosis. Effect of anti-miR-93 and PTEN expression vector on PTEN protein expression and its downstream factors in Saos cells after 48 hours of transfection
Fig. 5
Fig. 5
Effects of anti-miR-93 and PTEN expression vectors on the cell cycle distribution in Saos. Cells were treated and analyzed by flow cytometry after staining with PI (a-d). Histogram shows quantitative percentage of diploid cells (DNA content) in each cell cycle phase (e). Effects of anti-93-miR and PTEN vector on induction of apoptosis in Saos. Western blot showed the expression of PARP and its cleaved form (f). Transfection of anti-93-miR and PTEN plasmid with low dose Adriamycin showed the expression of PARP and its cleaved form Saos (g). The cells were labelled with FITC annexin V and PI in oligo transfection (h) and oligo transfection with low dose Adriamycin (i). Each quadrants represent viable cells (Lower Left quadrant), early apoptotic cells (Lower Right), late or secondary necrotic cells (Upper Right), and primary necrotic cells (Upper Left), respectively
Fig. 6
Fig. 6
Anti-miR-93 and PTEN vector suppressed in vivo tumor growth. Four groups were; (1) untreated (n = 7); (2) transfected with of negative control-miRs (n = 7); (3) transfected with anti-miR-93 (n = 7); and (4) transfected with PTEN expression vector (n = 7). Tumor volumes were measured at the time points indicated after the tumor cell inoculation. (**) P < 0.01. Immunofluorescence of PTEN (c) and p-Akt (f) following transfection of anti-miR-93 and PTEN vector into tumor cells. The number of PTEN (d) and p-Akt (f) positive cells per unit area in the tumor lesion
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