U2-OS cells and SAOS2 cells were purchased from the American Type Culture Collection (Manassas, VA, USA) and maintained in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 15% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) in a humidified atmosphere with 5% CO₂ at 37°C. For siRNA transfection, siRNA targeting Rb (Cell Signaling Technology Inc., Danvers, MA, USA) and Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific Inc.) were mixed and incubated for 20 min at room temperature. Then the mixture was added to cells plated in growth medium without antibiotics. The medium was changed after 4–6 h. For lentivirus infection, lentivirus targeting two different sequences was purchased from Shanghai GenePharma Co., Ltd. (Shanghai, China): KD1 targeting CGG CCT AAA GTA CCT TCA TAA and KD2 targeting CGG CCT AAA GTA CCT TCA TAA. Cells were plated in growth medium without antibiotics. At the time of infection, growth medium was replaced by medium containing lentivirus and polybrene (8 µg/ml; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and incubated for 12 h at 37°C.

Pretreated U2-OS cells and SAOS2 cells infected with shRBEL1-1, shRBEL-2 or scramble lentivirus were plated in 6-well plates (300 cells/well) and were maintained in DMEM supplemented with 15% FBS in a humidified atmosphere of 5% CO₂ at 37°C. Growth medium was changed every two days. After 12 days, cultured cells were fixed in 4% paraformaldehyde (Santa Cruz Biotechnology, Inc.) for 2 h and stained with 0.1% crystal violet (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) for 20 min. Colonies containing more than 50 individual cells were then counted with the inverted microscope (CKX41; Olympus Corporation, Beijing, China).

Cell proliferation was determined using a Cell Proliferation ELISA kit (containing BrdU labeling solution, FixDenat and anti-BrdU-POD; Roche Diagnostics GmbH, Mannheim, Germany). Briefly, pretreated U2-OS and SAOS2 cells were plated in a 96-well plate. The following day, 10 µl/well BrdU labeling solution was added and cells were incubated for 3 h. The medium was replaced by 200 µl/well FixDenat and incubated for 30 min at room temperature. Then FixDenat was removed using 100 µl/well anti-BrdU-POD. After washing twice with washing buffer, 100 µl/well substrate solution was added. The absorbance was measured at 450 nm with the ELx808 Absorbance Reader (Bio-Tek Instruments, Inc., Winooski, VT, USA).

Pretreated U2-OS and SAOS2 cells were harvested and washed twice in phosphate-buffered saline (PBS). Then cells were fixed in 70% ethanol at −20°C for 4 h. Cells were washed twice with PBS and stained with propidium iodide (PI)/RNase Staining Buffer (BD Biosciences, Franklin Lakes, NJ, USA) for 15 min. Cell cycle distribution was then analyzed by flow cytometry with the FACSCalibur flow cytometer (BD Biosciences).

Total RNA was extracted using TRIzol reagent (Invitrogen, Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Reverse transcription was performed using RevertAid First Strand cDNA Synthesis kit (Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Real-time PCRs were conducted using an ABI Prism 7500 Detection system (Applied Biosystems, Foster City, CA, USA) and SYBR Select Master mix system (Applied Biosystems) according to the manufacturer's instructions. Briefly, 10 µl 2X SYBR Select Master Mix was mixed with primers (200 nM), cDNA template (100 ng) and RNase free water made up to 20 µl. The cycling conditions werw as follows: 50°C for 2 min and 95°C for 2 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min.

Whole-cell extracts were prepared in radioimmunoprecipitation assay buffer supplemented with 1 mM phenylmethylsulfonyl fluoride (Beijing Solarbio Science & Technology Co., Ltd.). Total protein concentration was resolved by 10% SDS-PAGE (Beijing Solarbio Science & Technology Co., Ltd.). The proteins were then transfered to polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). Immunoblot analysis was conducted with incubation overnight at 4°C with the following antibodies: Anti-RBEL1 (1:1,000; ab111866) from Abcam (Cambridge, MA, USA), anti-Rb (1:200; 554144) and anti-active Rb (1:200; 554164) from (BD Biosciences), anti-c-Myc (1:100; sc-789), anti-cyclin D1 (1:200; sc-25765), anti-cyclin A2 (1:200; sc-53234) and anti-cyclin-dependent kinase 2 (CDK2; 1:200; sc-748) from Santa Cruz Biotechnology Inc., and polyclonal rabbit anti-human tubulin (1:2,000; T2200) from (Sigma-Aldrich, St. Louis, MO, USA). Membranes were then incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit immunoglobulin G secondary antibodies (1:5,000; ZB-2301; Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China) and was detected using the Western Chemiluminescent HRP Substrate (EMD Millipore).

All data sets were analyzed by Student's t-test. Data are presented as the mean ± standard deviation from three independent experiments. Statistical analysis was conducted using Graphpad Prism software, version 6 (GraphPad Software, Inc., San Diego, CA, USA). P<0.05 was considered to indicate a statistically significant difference.

To explore the potential role of RBEL1 in osteosarcoma, its expression in U2-OS and SAOS2 osteosarcoma cell lines was inhibited by shRNA lentivirus targeting two different sequences. Knockdown efficiency was confirmed by RT-qPCR and immunoblot analysis (Fig. 1A and B). Then RBEL1 knockdown cells and negative control cells were subjected to a colony formation assay. As shown in Fig. 1C, colony number and size were suppressed following RBEL1 knockdown. Furthermore, a BrdU incorporation assay was performed to determine whether RBEL1 depletion also had an impact on cell proliferation. In addtion, in U2-OS and SAOS2 cells RBEL1 depletion significantly inhibited BrdU incorporation suggesting suppressed proliferation (Fig. 1D). These data suggest that RBEL1 may act as a tumor suppressor in osteosarcoma.

The cell cycle of eukaryotic cells is conventionally divided into four phases and requires three switch-like transitions at the onset of S phase and at entry and exit of mitosis. Among them, G1-S transition is the most deregulated cell cycle control mechanism in malignant diseases (10). Thus it was hypothesized that RBEL1 may modulate G1-S transition thereby promoting cell proliferation in osteosarcoma cells. Using flow cytometry, cell cycle distribution of U2-OS and SAOS2 cells infected with RBEL1 shRNA or negative control shRNA was examined. As shown in Fig. 2, RBEL1 depletion in U2-OS cells resulted in enhanced G1-S arrest compared with negative control cells. Similar results were also shown in SAOS2 cells. These results indicate that RBEL1 may participate in G1-S transition regulation in osteosarcoma.

Rb is the major player in cell cycle control (11,12). A recent study indicated that RBEL1 negatively regulates Rb activation (8). Therefore, it was hypothesized that RBEL1 may regulate G1-S transition by inhibiting Rb activation. The impact of RBEL1 depletion on Rb expression was analyzed. An immunoblot assay showed that following RBEL1 knockdown, osteosarcoma cells exhibited increased expression of Rb compared with control cells (Fig. 3A). As the hypophosphorylated form of Rb is the active form that suppresses cell proliferation, it was examined whether hypophosphorylated Rb was also upregulated. As expected, RBEL1 knockdown cells expressed more hypophosphorylated Rb compared with the control (Fig. 3A). It is well-known that Rb targets the E2F transcription factor to suppress its downstream oncogene expression (11,12). To investigate whether RBEL1 depletion affects E2F transciptional activity, the expression of its downstream targets, such as cyclin A2, cyclin D1, c-Myc and CDK2, were examined. In agreement with above results, the mRNA and protein expression of these oncogenes was downregulated following RBEL1 knockdown in both cell types (Fig. 3B–D). These results indicate that RBEL1 may be a key regulator of Rb and its downstream targets.

The results indicated that RBEL1 modulates cell proliferation and G1-S transition, and can inhibit Rb activity. Thus it was investigated whether RBEL1 exerts its oncogenic effects by inhibiting Rb. To test this hypothesis, Rb expression was suppressed in RBEL1 knockdown cells to examine whether RBEL1 knockdown-induced tumor suppression can be rescued. The knockdown efficiency was confirmed by RT-qPCR and an immunoblot assay (Fig. 4A and B). A flow cytometry assay showed that in U2-OS and SAOS2, RBEL1 knockdown-induced G1-S arrest was decreased after Rb suppression (Fig. 4C and D). Furthermore, Rb suppression reversed colony formation capability in cells with RBEL1-knockdown (Fig. 4E). BrdU incorporation was also increased following Rb suppression (Fig. 4F). These results suggest that RBEL1 exerts its oncogenic effects by inhibiting Rb.