MG-63, Saos-2, U2OS and HOS human osteosarcoma cells lines were obtained from American Type Culture Collection (Manassas, VA, USA). HEK293T cells were obtained from the Institute of Cell and Biochemistry Research of the Chinese Academy of Science (Shanghai, China). The cell lines were cultured with Dulbecco's modified Eagle's medium (DMEM; Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Invitrogen; Thermo Fisher Scientific, Inc.) in a humidified atmosphere containing 5% CO₂ at 37°C.

The mouse anti-human monoclonal antibodies targeting UHRF1 (cat no. ab57083; 1:2,000 dilution) and GAPDH (cat no. ab9484; 1:5,000 dilution) were purchased from Abcam (Cambridge, MA, USA). Human Rb1-specific rabbit polyclonal antibody (cat. no. 9313; 1:2,000 dilution) was purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). The mouse anti-human E-cadherin monoclonal antibody (cat no. sc-21791; 1:2,000 dilution) was obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). The pLenti-UHRF1 lentiviral expression vector and the control vector were purchased from OriGene Technologies, Inc. (Beijing, China). PLKO.1 lentiviral vectors containing short hairpin (sh)RNA inserts against Rb1 were purchased from Sigma-Aldrich (St. Louis, MO, USA). The target sequences were as follows: shRb-A, 5′-GCCTTTGATTCGTTCCTTCTT-3′ and shRb-B, 5′-TGTGAAATACTGGCCCGAGAA-3′.

Transfection of the cells was performed using FuGENE transfection reagent (Roche Diagnostics, Indianapolis, IN, USA), according to the manufacturer's protocol (3.5×10⁶ cells in a 10 cm dish with 20 µl FuGENE). The lentiviral expression vector containing the pLenti-UHRF1/PLKO.1 or the control/empty vector were transfected into the HEK293T cells. The recombinant lentivirus was subsequently harvested, filtered through Millipore Millex-HV 0.45 µM polyvinylidene difluoride filters (Millipore, Billerica, MA, USA) and transduced into the target cells (MG-63, Saos-2 and U2OS cells at 60% confluence) with 8 µg/ml polybrene (Sigma-Aldrich). After 48 h of incubation, the cells were selected with fresh puromycin-containing media (2.0 µg/ml; Sigma-Aldrich). Following puromycin selection for 48 h, the expression levels of UHRF1 and Rb1 were quantified using western blot analysis.

Cell proliferation was determined using a CCK-8 assay (Dojindo, Kumamoto, Japan). Briefly, the cells were plated in 96-well plates at 2,500 cells/well and cultured in DMEM. Every 24 h, 10 µl CCK-8 was added to each well containing 100 µl DMEM once. The plates were then incubated for a further 2 h at 37°C. The cell growth was monitored every 24 h for 7 days using the CCK-8 assay. Absorbance was measured at 450 nm using a microplate reader (Infinite Pro 2000; Tecan GmbH, Grödig, Austria).

To assess the role of UHRF1 in cell invasion, a total of 1×10⁵ cells were suspended in 100 µl DMEM supplemented with 10% FBS, and were seeded into the upper compartment of Matrigel-coated (BD Biosciences, Franklin Lakes, NJ, USA) Transwell chambers (24-well; 8 µm; Merck Millipore, Darmstadt, Germany). The cells were incubated for 24 h at 37°C in a 5% CO₂ chamber. The cells, which did not invade through the pores were removed using a cotton swab. The cells on the lower surface of the membrane were stained with a hematoxylin and eosin staining kit (Baixu of Biotechnology, Shanghai, China) and counted with a microscope (Leica DM 5000 B; Leica Microsystems, Wetzlar, Germany). Results are presented as the average number of cells in five randomly selected fields.

The cells were suspended in lysis buffer (Beyotime Institute of Biotechnology, Nanjing, China) containing a mixture of protease and phosphatase inhibitors (both from Roche Diagnostics GmbH, Mannheim, Germany). The cell lysates were then centrifuged at 11,500 × g for 10 min at 4°C. Protein concentrations were estimated using the Quick Start™ Bradford Protein Assay (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The samples were separated by SDS-PAGE (4–29% gradient; Bio-Rad Laboratories, Inc.) and then blotted onto a polyvinylidene difluoride membrane (Merck Millipore). The membranes were blocked with 5% non-fat dried milk in Tris-buffered saline with 1% Tween 20 (TBST; Sigma-Aldrich) for 1 h at room temperature prior to incubation with specific primary antibodies overnight at 4°C. Following three washes with TBST, the membrane was incubated with peroxidase-conjugated secondary antibodies [anti-mouse immunoglobulin (Ig)G (cat no. 7076; 1:5,000 dilution) and anti-rabbit IgG (cat no. 7074) (both from Cell Signaling Technology, Inc.)] for 30 min at 37°C and then washed three times with TBST. The bound antibodies were detected using chemiluminescent horseradish peroxidase substrate (Merck Millipore) and images were captured using an LAS-4000 digital imaging system (GE Healthcare Life Sciences, Little Chalfont, UK).

Values are expressed as the mean ± standard deviation. GraphPad 6.01 Prism software (GraphPad, Inc., La Jolla, CA, USA) was used for statistical analyses. Each experiment was repeated three times. P<0.05 was considered to indicate a statistically significant difference.

UHRF1 has been reported to be overexpressed in various types of cancer (13). The present study examined the expression levels of UHRF1 in four human osteosarcoma cell lines. As shown in Fig. 1A, the expression of UHRF1 was detected in all of the cell lines. Overexpression of UHRF1 can increase cell proliferation (14,21), therefore, the effects of UHRF1 on the proliferation of human osteosarcoma cells were also investigated. UHRF1 was stably overexpressed in the MG-63 and Saos-2 osteosarcoma cell lines (Fig. 1B). As expected, overexpression of UHRF1 increased the proliferation rate of the MG-63 and Saos-2 osteosarcoma cell lines (Fig. 1C).

In order to investigate the function of UHRF1 on the invasion of osteosarcoma cells, Transwell invasion assays were used with three human osteosarcoma cell lines in vitro. The overexpression of UHRF1 significantly increased the invasion of MG-63 and U2OS human osteosarcoma cells (Fig. 2). By contrast, the overexpression of UHRF1 had no significant effect on the invasion of the Saos-2 cells (Fig. 2). Homozygous deletion of the Rb1 gene has been identified in Saos-2 cells (22,23), whereas MG-63 and U2OS cells exhibit normal expression levels of Rb1 (23,24). Therefore, the present study hypothesized that Rb1 may be involved in regulating the invasion of osteosarcoma cells by UHRF1.

To test the hypothesis that Rb1 may be involved in the regulation of the invasion of osteosarcoma cells by UHRF, the expression levels of Rb1 were quantified in the osteosarcoma cell lines. As expected, the MG-63 and U2OS cells exhibited normal expression levels of Rb1. The expression of Rb1 in the Saos-2 cells containing the homozygous deletion of Rb1 was not detected (Fig. 3A). To further investigate the mechanism underlying the effect of UHRF1 on the regulation of osteosarcoma cell invasion, the expression of Rb1 was stably knocked down in the MG-63 cells (Fig. 3B). The stable known-down of Rb1 resulted in UHRF1 being overexpressed in the MG-63 cells (Fig. 3C). Following the knockdown of Rb1 in the Rb1-positive MG-63 cells, the invasion of the cells increased. By contrast, the indiced overexpression of UHRF1 had no effect on the invasion of the MG-63 cells following stable Rb1 knockdown (Fig. 3D). These results indicated that Rb1 appeared to be responsible for the regulation of cell invasion by UHRF1.

UHRF1 can inhibit the expression levels of Rb1 at the protein and mRNA levels (6). Furthermore, the inhibition of Rb1 suppresses the expression of E-cadherin and increases EMT (8). The present study hypothesized that UHRF1 may inhibit the expression of Rb1 and thereby suppress the expression of E-cadherin. E-cadherin is known to suppress the invasion of cancer cells (25). Therefore, the UHRF1-mediated promotion of invasion may be the result of E-cadherin inhibition. The results of the present study demonstrated that the expression levels of E-cadherin were significantly lower in the Saos-2 cells, compared with the other Rb1-positive cells (Fig. 4A). Furthermore, overexpression of UHRF1 in the MG-63 and U2OS cells inhibited the expression of Rb1 and E-cadherin (Fig. 4B). The loss of E-cadherin is the initial or primary cause for EMT (26). In the present study, the UHRF1-overexpressing MG-63 cells exhibited marked changes in cell morphology, with transformation of the cobblestone-like epithelial cells to an elongated fibroblast-like morphology, and with pronounced cellular scattering that indicated EMT (Fig. 5). Similarly, knockdown of Rb1 in the MG-63 cells also showed EMT characteristic patterns (Fig. 5). However, UHRF1 had no effect on the cell morphology of the Saos-2 cells with the Rb1 deletion (Fig. 5). These results further supported the hypothesis that UHRF1 inhibits the expression of E-cadherin and promotes EMT through the suppression of Rb1.