Cell culture and transfection
The human embryonic kidney 293T cells, human non-cancerous osteoblast hFOB 1.19 cells and osteosarcoma cell lines (SaoS-2, HOS, U-2OS and MG-63) were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). The cells were cultured in Eagle’s minimum essential medium or McCoy’s 5A medium (Gibco Life Technologies, Grand Island, NY, USA), supplemented with 10% (v/v) fetal bovine serum (FBS; Gibco, Rockville, MD, USA) and 100 U/mL penicillin and 100µg/mL streptomycin (Sigma-Aldrich, St, Louis, MO, USA) at 37℃ in a humidified incubator with 5% CO2. MiR-383 mimics, miR-383 inhibitors, PSMB5 siRNA and their negative controls (NC) were purchased from GenePharma (Shanghai, China). Cell transfection was performed using the TurboFect (ThermoFisher Scientific) according to the manufacturer’s instructions. Plasmids or oligonucleotides were used at a final concentration of 2µg or 50nM.
Osteosarcoma tissue samples
Normal bone tissue and osteosarcoma tissue chip (L714901, 70 cases of osteosarcoma and 1 normal sample) were obtained from the Xi 'an Zhongke Guanghua Intelligent Biotechnology.
RNA isolation and quantitative Real-time PCR
RNAiso Plus Reagent (TaKaRa Biotechnology, Dalian, China) was selected to extract total RNA from the cultured cells according to the manufacturer’s instructions. Complementary DNA (cDNA) was synthesized using the Multiscript RT Kit (TaKaRa). After mixing the templates, primer pairs and SYBR Master Mixture (TaKaRa) gently, quantitative real-time PCR was performed on BIO-RAD CFX 96 detection system. PCR conditions were strictly controlled as follows: 3 min at 95℃ followed by 40 cycles of 95℃ for 10s and 55℃ for 30s. The fold changes of mRNA and miR-383 expression levels were calculated as 2- ∆∆Ct. GAPDH served as an endogenous control for PSMB5 expression. U6 was used as an internal control for miR-383 expression.
Real-time PCR was carried out with the following primers:
miR-383 RT:5’- GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACT
GGATACGACAGCCAC − 3’;
U6 RT: 5’ - AACGCTTCACGAATTTGCGT − 3’
miR-383: 5’- GTGCAGGGTCCGAGGT − 3’ (forward)
5’ - AGATCAGAAGGTGATTGTGGCT − 3’ (reverse);
PSMB5: 5’- CCATGGGCACCATGATCT − 3’ (forward)
5’ - GAAGGTGGCCCCTGAAAT − 3’ (reverse);
GAPDH: 5’ - TGTCCGTCGTCCATCTGA − 3’(forward)
5’ - CCTGCTTCACCACCTTCTT − 3’(reverse)
U6: 5’- CTCGCTTCGGCAGCACA − 3’ (forward)
5’ - AACGCTTCACGAATTTGCGT − 3’ (reverse);
Western blot
Proteins were extracted from cells using RIPA lysis buffer, and the concentration was measured using the Bradford Assay kit (Beyotime Institute of Biotechnology) according to the manufacturer’s instructions. A total of 20 µg of protein was separated by 10% SDS-PAGE electrophoresis and transferred onto nitrocellulose membranes. After blocking with 5% nonfat milk in Tween-Tris-buffered saline (TBST), the membranes were incubated with primary antibodies against PSMB5 or GAPDH (Cell Signaling Technology, San Jose, CA, USA)) at 4℃ overnight. Subsequently, the membranes were washed and incubated with horseradish peroxidase-conjugated goat anti-mouse or -rabbit IgG (1:5000; Cell Signaling Technology, San Jose, CA, USA) for 1 hour at room temperature. The target protein was visualized by enhanced SuperSignal WestPico chemiluminescent (Pierce, Rockford, IL, USA), and the relative protein expression levels were evaluated using Quantity One software.
Proteasome activity assay
The proteasome activity was measured by the Proteasome-GloTM cell-based assays according to the manufacturer’s instructions (Promega, USA). Briefly, luciferin detection reagent and proteasome-Glo substrate were mixed in cell-based buffer and incubated at room temperature for 30 min before being added to the samples. The luminescence was determined using a Turner Biosystems luminometer (Turner Biosystems, Sunnyvale, CA) after a 10–20 min reaction. In some experiments, cells were transfected with miR-383 mimics/NC for 48 h or/and treated with bortezomib for 24 h.
Cell proliferation assay
Cells were seeded into 96-well plates at a density of 5×103 cells/well in septuple in 100 µL of medium and cultured. After incubation overnight, cells were subjected to DMSO/bortezomib (5nM or 10nM). After treatment for another 3 d, 10 µL of CCK-8 was added into each well. After 1 h of incubation, the absorbance at 450 nm was measured using a microplate reader (iMark; Bio-Rad Laboratories, Inc., Hercules, CA, USA). The assays were repeated three times. In some experiments, cells were transfected with miR-383 mimics/NC for 48 h or/and treated with bortezomib for 24 h.
Dual-luciferase reporter assay
The 3’-UTR luciferase reporter constructs were made by cloning PSMB5 3’-UTR sequence (WT) which was predicted as a binding site for miR-383 or its mutant sequence (MT) into pMir-Glo vector at EcoRI and XhoI sites. To determine if miR-383 can bind to PSMB5 3’-UTR, the control pMir-Glo vector, WT or MT PSMB5 were co-transfected with NC, miR-383 mimics or miR-383 inhibitor into 293T cells. Luciferase activity was measured at 48 hours of post-transfection by the classical Dual-Luciferase Reporter Assay System (Promega). The firefly luciferase signal was normalized to the renilla luciferase signal and expressed as relative luminescence units (RLU).
Caspase-3 activity assay
Cells were transfected with PSMB5/NC -siRNA for 48 h and/or treated with bortezomib for 24 h. After treatment, apoptosis was measured by a caspase-3 activity assay kit (Promega). Briefly, cells were washed twice with PBS and suspended in caspase lysis buffer (containing 1M Tris-HCl, 2mM MgCl2, 150mM NaCl, and 10mM DTT) for 15 min on ice. The clear supernatant was collected after the cells were centrifuged. Assay buffer containing caspase-3 substrate was then added to the supernatant. The incubation was performed for 1 h with readings recorded at 5-min intervals. Fluorescence was measured at 360 nm excitation and 460 nm emission wavelengths. Values were normalized to protein concentration and expressed as fold change of activity relative to DMSO control.
Cell flow cytometry apoptosis assay
Cell apoptosis was also evaluated by flow cytometry using an Annexin-V-FITC Apoptosis Detection Kit (KeyGen Biotech Co. Roche) according to the manufacturer’s instructions. Briefly, the treated cells were harvested, washed and resuspended in 195µL of Annexin V-FITC binding buffer. Then a volume of 5 µL of Annexin-V-FITC was added and mixed. The cells were stained in the dark for 10 min at room temperature. After that, cells were centrifuged at 1000g for 5 min and resuspended in 190µL of Annexin V-FITC binding buffer and 10µL of PI was added and mixed gently, and the cells were kept on ice in the dark and immediately subjected to flow cytometry.
Fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC)
FISH was performed to detect miR-383 expression in human osteosarcoma and normal bone tissues, which was conducted as described previously [28]. The miR-383 probe 5‘-AGCCACAATCACCTTCTGATCT-3’ was synthesized. A semi-quantitative scoring criterion was used for FISH, where the staining intensity was recorded. Protein expression levels of PSMB5 from the osteosarcoma tissue were determined by standard immunohistochemistry protocols. Sections were scanned, and the images were then digitalized. Image J software was used to calculate the integrated optical density (IOD) of the indicated mRNAs and proteins.
Statistical analysis
Statistical analysis was performed using the SPSS 13.0 software (IBM, Armonk, NY, USA). Student t-tests were carried out to make a comparison between different groups. Data were expressed as mean ± SD. A value of p༜0.05 was considered statistically significant.