OS cell lines Saos-2, U-2 OS, HOS and MG-63 and the normal osteoblast cell line NHOst were obtained from the American Type Culture Collection (ATCC; Rockville, MD, USA). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS) (both from Life Technologies, Carlsbad, CA, USA) at 37°C in a humidified incubator containing 5% CO₂.

Total RNA was extracted using the miRNA Isolation kit (Life Technologies) according to the manufacturer’s instructions. For detection of miR expression, the MicroRNA reverse transcription kit (Life Technologies) was used to convert 10 ng of total RNA into cDNA, according to the manufacturer’s instructions. Real-time PCR was then performed using the miRNA Q-PCR detection kit (GeneCopoeia, Rockville, MD, USA) on Applied Biosystems 7500 Real-Time PCR System. U6 gene was used as an internal reference. The relative miR-204 expression was normalized to U6. The relative expression was analyzed by the 2^−ΔΔCt method.

Cells were lysed with ice-cold lysis buffer (50 mM Tris-HCl, pH 6.8, 100 mM 2-ME, 2% w/v SDS, 10% glycerol). After centrifugation at 20,000 x g for 10 min at 4°C, proteins in the supernatants were quantified and separated with 10% SDS-PAGE. Then, proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane (Amersham Biosciences, Buckinghamshire, UK), which was then incubated with phosphate-buffered saline (PBS) containing 5% milk overnight at 4°C. The PVDF membrane was incubated with rabbit anti-Sirt1 monoclonal antibody (1:100) and rabbit anti-GAPDH monoclonal antibody (1:200) at room temperature for 3 h, respectively, and then with HRP-linked goat anti-rabbit secondary antibody (all from Abcam, Cambridge, UK) at room temperature for 1 h. The Thermo Scientific SuperSignal West Pico Chemiluminescent Substrate kit (Pierce, Rockford, IL, USA) was then used to detect the signals according to the manufacturer’s instructions. The relative protein expression was analyzed by Image-Pro Plus software 6.0, represented as the density ratio vs. GAPDH.

Lipofectamine 2000 (Life Technologies) was used to perform cell transfection following the manufacturer’s instructions. For functional analysis, Saos-2 and U-2 OS cells were transfected with scramble miR, miR-204 mimics, Sirt1 siRNA (all from Life Technologies) or co-transfected with miR-204 mimics and the Sirt1 plasmid (Nlunbio, Changsha, China), respectively.

We screened the target genes of miR-204 using TargetScan (http://www.targetscan.org/index.html), PicTar (http://pictar.bio.nyu.edu/) and miRanda (http://www.microrna.org/microrna/home.do).

Luciferase reporter assay was performed to clarify whether Sirt1 is a direct target gene of miR-204. Briefly, total cDNA from OS Saos-2 cells was used to amplify the 3′-UTR of Sirt1, which was then cloned into the pMIR-REPORT vector (Life Technologies). Mutations were introduced within the potential seed sequences of the 3′-UTR of Sirt1 using the QuikChange Site-Directed Mutagenesis kit (Stratagene). The seed sequences AAAGGGAA were mutated into AAACCCAA. Using Lipofectamine 2000, the cells were transfected with the pMIR-REPORT vectors containing the wild-type or mutant type of Sirt1 3′-UTR and miR-204 mimics, respectively. The pRL-Sv40 vector (Promega, USA) carrying the Renilla luciferase gene was used as an internal control. Luciferase activity was determined after 48 h using the Dual-Glo substrate system and LD400 luminometer (Beckman Coulter, Kraemer Boulevard, Brea, CA, USA). Data are presented as the ratio of Renilla luciferase to firefly luciferase.

The MTT assay was used to measure cell proliferation. At 48 h post-transfection, the transfection medium in each well was replaced with 100 µl of fresh serum-free medium containing 0.5 g/l MTT. Subsequent to incubation at 37°C for 4 h, the MTT medium was removed by aspiration, and 50 µl of dimethyl sulfoxide was added to each well. Following incubation at 37°C for a further 10 min, the optical density at 570 nm was measured using the BioTek™ EL×800™ Absorbance Microplate Reader (BioTek, Winooski, VT, USA).

A wound healing assay was performed to evaluate the cell migratory capacity of the OS cells in each group. In brief, OS cells were cultured to full confluency. Wounds of ~1 mm in width were created with a plastic scriber, and cells were washed and incubated in a serum-free medium. After wounding for 24 h, the cells were incubated in a medium including 10% FBS. After being cultured for 48 h, the cells were fixed and observed under a microscope.

Cells in each group were starved in serum-free medium for 24 h, and then resuspended in serum-free medium. The cell suspension was added into the upper chamber, while the lower chamber was filled with base medium containing 10% FBS. After incubation for 24 h, the cells that attached to the bottom were stained with crystal violet for 20 min, and then washed and dried in air. Invasive cells were observed under a microscope.

Data are expressed as mean ± standard deviation from at least three separate experiments. Differences were analyzed using one-way analysis of variance (ANOVA). SPSS 18.0 software was used to perform statistical analysis. P<0.05 was considered to indicate a statistically significant result.

Real-time RT-PCR was used to detect the expression of miR-204 in four OS cell lines Saos-2, U-2 OS, HOS and MG-63, and in the normal osteoblast cell line NHOst. Our data showed that the expression of miR-204 was significantly reduced in the OS cell lines, when compared with that in the normal osteoblast cell line NHOst (Fig. 1). We then chose Saos-2 and U-2 OS cells to perform functional analysis of miR-204 in OS in vitro.

The role of miR-204 in the regulation of proliferation, migration and invasion of OS cells was further investigated. After transfection of the Saos-2 and U-2 OS cells with miR-204 mimics, we firstly determined the level of miR-204 in each group. As shown in Fig. 2A, the miR-204 level was significantly increased after transfection compared to the control group. Cell proliferation assay data showed that overexpression of miR-204 notably inhibited the proliferation of the Saos-2 and U-2 OS cells, when compared to the control groups (Fig. 2B). Subsequently, we determined the cell migration by performing a scratch assay. As shown in Fig. 3A, the migratory capacity of the OS cells transfected with the miR-204 mimics was significantly reduced, when compared to this capacity in the control groups. Moreover, miR-204 overexpression also suppressed the invasive capacity of the Saos-2 and U-2 OS cells, when compared to the control groups (Fig. 3B). These findings suggest that miR-204 plays an inhibitory role in the regulation of proliferation, migration and invasion of OS cells.

According to bioinformatics predication, Sirt1 is a putative target gene of miR-204 (Fig. 4A). To confirm that Sirt1 is a target gene of miR-204, the luciferase reporter assay was performed to clarify whether miR-204 directly binds to their seed sequences in the Sirt1 3′-UTR in the Saos-2 cells. The luciferase activity was significantly decreased in the Saos-2 cells co-transfected with the wild-type (WT) Sirt1 3′-UTR and miR-204 mimics, but showed no difference in the Saos-2 cells co-transfected with the mutant type (MUT) Sirt1 3′-UTR and miR-204 mimics, when compared with that in the control groups (Fig. 4B). These data indicate that Sirt1 is a target gene of miR-204. We then detected the protein level of Sirt1 in the Saos-2 and U-2 OS cells with or without transfection with the miR-204 mimics. As shown in Fig. 4C, overexpression of miR-204 inhibited the protein level of Sirt1 in the OS cells when compared to the control groups, indicating that miR-204 negatively mediates the protein expression of Sirt1 through directly binding to the 3′-UTR in Sirt1 mRNA.

To further clarify whether Sirt1 acts as a downstream effector in miR-204-mediated proliferation, migration and invasion of OS cells, we transfected Saos-2 and U-2 OS cells with Sirt1 siRNA or co-transfected the cells with miR-204 mimics and the Sirt1 plasmid. After that, we firstly determined the protein expression of Sirt1 by performing western blotting in each group. As shown in Fig. 5A, transfection with Sirt1 siRNA significantly inhibited the protein expression of Sirt1 in the OS cells when compared to that in the control groups, while transfection with the Sirt1 plasmid reversed the inhibitory effect of miR-204 overexpression on Sirt1 protein expression in the OS cells. Subsequently, we determined the proliferation, migratory and invasive capacities of the OS cells in each group. Our data showed that inhibition of Sirt1 significantly inhibited OS cell proliferation (Fig. 5B), migration (Fig. 6A) and invasion (Fig. 6B). However, overexpression of Sirt1 reversed the inhibitory effect of miR-204 upregulation on OS cell proliferation, migration and invasion (Figs. 5B, and 6A and B), suggesting that Sirt1 is involved in the miR-204-mediated proliferation, migration and invasion of OS cells.

As EMT is also a key step in cancer metastasis in addition to migration and invasion, we further investigated the roles of miR-204 and Sirt1 in the EMT of OS cells. The protein expression of N-cadherin and E-cadherin was detected in the OS cells transfected with miR-204 mimics or Sirt1 siRNA using western blotting. As shown in Fig. 7, the protein expression of E-cadherin was increased, while the N-cadherin protein level was decreased after miR-204 overexpression or Sirt1 knockdown in the OS cells, suggesting that miR-204 plays a suppressive role of EMT in OS cells via targeting Sirt1.