Experimental animals and OA model
Male Sprague–Dawley rats (200–250 g) were obtained from Henan Research Center of Laboratory Animal (Zhengzhou, China). The rats were anesthetized by 30 mg/kg pentobarbital sodium, and destabilization of the medial meniscus (DMM) was performed as previously described [14]. Briefly, in DMM group (n = 10), after the right knee joint was exposed by a medial capsular incision, the extensor muscles were gentle moved and the medial meniscus was transected, then the medial capsular incision and the skin were seamed. In sham group (n = 10), the joint capsule was opened but the medial meniscotibial ligament was left intact. After 4 weeks, the rats were killed and cartilage tissues were harvested under sterile conditions. All animals were treated according to the national guidelines of the care and use of laboratory animals with the approval of the Ethics Committee for Animal Research.
Cell isolation and cell culture
Chondrocytes were isolated and cultured as previously described [15]. In brief, rat cartilage tissues were cut into small pieces and digested with trypsin (Sigma-Aldrich, St. Louis, MO, USA). After digested into monolayer cells, chondrocytes were seeded into culture plate (Corning, Toledo, NY, USA) in DMEM medium (Gibco, Rockville, MD, USA) with 10% FBS (Gibco), 100 U/ml penicillin (Gibco), 100 mg/ml streptomycin (Gibco) at 37 °C. Adherent chondrocytes at 70–80% confluency were cultured in serum-free medium for 12 h, and then stimulated with 10 ng/ml IL-1β for 2 h to mimick OA chondrocytes. HEK 293T cells were purchased from American Tissue Culture Collection (ATCC, Manassas, VA, USA), which was cultured in MEM medium (Gibco) with 10% FBS.
Cell transfection
The MEG3 and SMAD7 sequences were synthetized from Sangon Biotech (Shanghai, China) and cloned into a pcDNA3.1 plasmid (Thermo Fisher Scientific, Waltham, MA, USA) to construct MEG3 overexpression vector (MEG3) and SMAD7 overexpression vector (SMAD7). All siRNAs (si-MEG3, si-SMAD7, si-NC), miRNAs mimics (miR-16 mimics, miR-NC), and miRNA inhibitors (anti-miR-16, anti-miR-NC) were also obtained from Sangon Biotech. Plasmids or oligonucleotides were transfected into IL-1β-induced chondrocytes by using the Lipofectamine 3000 transfection reagent (Life Technologies, Carlsbad, CA, USA) according to the protocols of manufacturer.
RNA extraction and RT-qPCR
Total RNA was isolated from cartilage tissues of rat OA model and treated chondrocytes using GenElute™ Total RNA Purification Kit (Sigma-Aldrich) referring to the instructions of manufacturer. 500 ng total RNA was used to detected the relative MEG3 and miR-16 expression by using QuantiNova SYBR Green PCR kit (Qiagen, Hilden, Germany) on an 7500 fast real-time PCR system (Applied Biosystems, Waltham, MA, USA). GAPDH or U6 was used as internal reference and the 2−ΔΔCt method was used to calculate the expression. For the RT-qPCR analysis, the following primers were used: MEG3: 5′-CTGCCCATCTACACCTCACG-3′ (forward) and 5′-CTCTCCGCCGTCTGCGCTAGGGGCT-3′ (reverse); miR-16: 5′-TAGCAGCACGTAAATATTGGCG-3′ (forward) and 5′-TGCGTGTCGTGGAGTC-3′ (reverse); GAPDH: 5′-TGCACCACCAACTGCTTAGC-3′ (forward) and 5′-GGCATGCACTGTGGTCATGAG-3′ (reverse); U6: 5′-GCTTCGGCAGCACATATACTAAAAT-3′ (forward) and 5′-CGCTTCACGAATTTGCGTGTCAT-3′ (reverse).
Cell viability assays
Cell viability of treated chondrocytes was assessed by Cell Counting Kit-8 (CCK-8, Sigma-Aldrich) referring to the instructions of manufacturer. Briefly, IL-1β-induced chondrocytes were transfected for 24, 48 and 72 h, then 10 μl of CCK-8 solution was added to each well and incubated for 2 h. Subsequently, the absorbance at 450 nm was measured using a microplate reader (Bio-Rad Laboratories, Hercules, CA, USA).
Flow cytometry
The apoptosis of treated chondrocytes was determined by flow cytometry assay with Annexin V-FITC Apoptosis Detection Kit (Abcam, Cambridge, UK). The apoptotic rate was analyzed with a flow cytometer (FACSCalibur, Becton–Dickinson, Franklin Lakes, NJ, USA) using CellQuest software.
Western blot analysis
Cells were completely lysed in 200 l of the lysis buffer (Takara, Dalian, China) and then centrifuged at 8000g for 5 min. Proteins were separated by 12% SDS-PAGE, and transferred to PVDF membranes (Millipore, Billerica, MA, USA). The membranes were blocked by 5% skimmed milk in TBS for 2 h. After washed three times by TBS containing 0.1% Tween-20 (TBST), the PVDF membranes were incubated with anti-Bax (Cell Signaing Technology, Danvers, MA, USA), anti-Bcl2 (Cell Signaing Technology), anti-SMAD7 (Cell Signaing Technology), anti-β-actin (Cell Signaing Technology) overnight at 4 °C, respectively. After washed with TBST, the PVDF membranes were incubated with HRP-conjugated secondary antibodies (Cell Signaling Technology). Lastly, the PVDF membranes were exposed to ECL Western Blotting Substrate (Solarbio, Beijing, China) for 5 min and were quantified using VersaDoc 4000MP imaging system (Bio-Rad).
RNA immunoprecipitation (RIP) assay
RNA immunoprecipitation assay was performed by Imprint RNA immunoprecipitation kit (Sigma-Aldrich) referring to the recommended protocols of manufacturer. Firstly, IL-1β-induced chondrocytes were collected and resuspended in RIP lysis buffer (Solarbio), subsequently centrifuged at 12,000g for 5 min. Then, cell lysate was incubated with anti-Argonaute2 (anti-Ago2) or anti-IgG (negative control) overnight at 4 °C, followed by the addition of Protein A magnetic beads to get the immunoprecipitation complex. Total RNA was isolated using GenElute™ Total RNA Purification Kit (Sigma-Aldrich). Lastly, the relative enrichment of MEG3 and miR-16 were determined by RT-qPCR analysis.
Luciferase reporter assay
The partial squences of MEG3 and 3′-UTR of SMAD7 containing the putative binding sites of miR-16 were synthetized and obtained from Sangon Biotech (Shanghai), then were cloned into the pmirGLO Dual-Luciferase miRNA Target Expression Vectors (Promega, Madison, WI, USA) to construct wild-type reporter vectors MEG3 (WT) and SMAD7 (WT), respectively. The mutant MEG3 sequences and 3′-UTR of SMAD7 sequences containing the putative binding sites of miR-16 were performed by Q5 Site-Directed Mutagenesis Kit (New England Biolabs, Ipswich, MA, USA) and then cloned into pmirGLO vectors respectively, to construct mutant-type reporter vectors MEG3 (MUT) and SMAD7 (MUT). The MEG3 (WT) or MEG3 (MUT) were transfected into HEK 293T cells together with miR-NC, miR-16 mimics, anti-miR-NC or anti-miR-16. Similarly, the SMAD7 (WT) or SMAD7 (MUT) were transfected into HEK 293T cells together with miR-NC or miR-16 mimics. HEK 293T cells were contransfected with SMAD7 (WT) and miR-NC, miR-16 mimics, miR-16 mimics + pcDNA, or miR-16 mimics + MEG3. The relative luciferase activity was analyzed by the Dual-Glo Luciferase Assay System (Promega).
Statistical analysis
Statistical analyses were preformed by Student’s t-test or one-way ANOVA using software SPSS 15.0 (SPSS Inc., Chicago, IL, USA). All data were presented as the mean ± standard deviation (mean ± SD). A P-value less than 0.05 was considered statistically significant.