The CHON-002 human chondrocyte cell line was purchased from the American Type Culture Collection (Manassas, VA, USA), and cultured in Dulbecco's Modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) with 10% heat-inactivated fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) and 0.1 mg/ml G-418 solution (Sigma-Aldrich; Merck-Millipore, Darmstadt, Germany).

TRIzol reagent (Thermo Fisher Scientific, Inc.) was used to extract RNA from chondrocytes (1×10⁷ cells), according to the manufacturer's instructions. This was followed by reverse transcription-polymerase chain reaction (RT-PCR) to amplify the coding region of GIT1. The primers used for GIT1 amplification are listed in Table I. The product was digested with KpnI and EcoRI restriction endonucleases (Takara Biotechnology Co., Ltd., Dalian, China), and then cloned into pcDNA3.1 vectors (Thermo Fisher Scientific, Inc.), sequenced and verified in a 3730xl DNA Analyzer (Applied Biosystems; Thermo Fisher Scientific, Inc.). Cells (1×10⁵) were seeded in 6-well culture plates and cultured until they reached ~70% confluence, before expression vectors were transfected using Lipofectamine 2000 transfection reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. The concentration of the GIT1 transfection vector used was 4 µg/well. The GIT1 small-interfering RNA (siRNA; cat. no. sc-35477; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), miR-195 mimics and inhibitors (Shanghai GenePharma Co., Ltd., Shanghai, China) were all transfected into cells at a concentration of 50 nM/well.

Total RNA was isolated from cultured cell samples using TRIzol reagent (Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions, and the mirVana miRNA Isolation kit (Applied Biosystems; Thermo Fisher Scientific, Inc.) was used to purify miRNAs. Target gene expression levels of GIT1 and miR-195 were measured using RT-qPCR. cDNA was synthesized by reverse transcription using random and oligo-dT primers (Promega Corporation, Madison, WI, USA) or specific primers for miRNA-195 (Takara Biotechnology Co., Ltd.), together with the GoScript Reverse Transcription System (Promega Corporation). qPCR was performed using the GoTaq qPCR Master Mix (Promega Corporation) and an ABI PRISM^® 7500 Sequence Detection System (Applied Biosystems; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. The thermocycling conditions were as follows: 95°C for 2 min; and 40 cycles of 95°C for 15 sec and 60°C for 32 sec. The primer sequences are shown in Table I. GAPDH served as a control for GIT1 expression and U6 as a control for miR-195. To measure miRNA expression, specific primers for miRNA-195 and U6 were used. Three independent experiments were conducted for each sample. Data were analyzed by comparing the 2^−ΔΔCq values (16).

Total protein was extracted by incubating cells (1×10⁶) in radioimmunoprecipitation assay (RIPA) buffer (cat. no. sc-24948; Santa Cruz Biotechnology, Inc.) for 10 min in an ice bath, prior to loading of 50 µg samples in 10% SDS-PAGE gels for separation and transferred to nitrocellulose membranes (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Membranes were blocked with 5% non-fat milk for 1 h at room temperature and then incubated with antibodies against GIT1 (cat. no. 2919; dilution, 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA) or GAPDH (cat. no. 2118; 1:1,000; Cell Signaling Technology, Inc.) in 5% non-fat milk overnight at 4°C. Immunoreactive proteins were visualized using incubation with horseradish peroxidase-conjugated IgG secondary antibodies (cat. no. 7074; dilution, 1:7,000–8,000; Cell Signaling Technology, Inc.) at room temperature for 1 h and enhanced chemiluminescence reagents (Pierce; Thermo Fisher Scientific, Inc.). Images were analyzed using Image-Pro Plus 6.0 (Media Cybernetics, Inc., Rockville, MD, USA). Each band was scanned with background correction, and values were expressed as the mean ± standard deviation.

The software applications miRanda (http://www.microrna.org/microrna/getGeneForm.do), TargetScanHuman (http://www.targetscan.org/vert_71/), miRBase (http://www.mirbase.org/) and miRWalk (http://zmf.umm.uni-heidelberg.de/apps/zmf/mirwalk2/) predicted that GIT1 has miR-195a-3p binding sites. The GIT1 3′-UTR was cloned into the psiCHECK-2 vector (Promega Corporation), and the seed region of the miR-195 binding site in the 3′-UTR was mutated using the QuikChange II Site-Directed Mutagenesis kit (Agilent Technologies, Inc., Santa Clara, CA, USA). For the luciferase assay, 2×10⁴ HEK293A cells (American Type Culture Collection) were seeded in 24-well dishes and were cultured until they reached 80% confluence. Cells were transfected with psiCHECK-2 (containing the wild-type GIT1 3′-UTR or the mutated form) together with miR-195 mimics using the Lipofectamine 2000 transfection agent (Invitrogen; Thermo Fisher Scientific, Inc.). Cells were analyzed at 24 h post-transfection. Firefly and Renilla luciferase activities were quantified in cell lysates using the Dual-Luciferase Reporter assay kit (Promega Corporation) on a Glomax 20/20 luminometer (Promega Corporation) according to the manufacturer's instructions. Luciferase readings were corrected to background readings and firefly luciferase values were normalized to Renilla values in order to determine the transfection efficiency. Samples were analyzed in triplicate and three independent experiments were conducted.

A total of 1×10⁷ HEK 293A cells were lysed in RIPA buffer containing 1 mM protease inhibitors (Roche Diagnostics GmbH, Mannheim, Germany) and centrifuged at 10,000 × g for 10 min at 4°C. For immunoprecipitation, 5 ml of supernatant from monoclonal anti-Ago2 antibody (cat. no. 2897; dilution, 1:50; Cell Signaling Technology, Inc.) was coupled to 80 µl Protein-G-Sepharose beads (GE Healthcare Life Sciences, Chalfont, UK). Beads were subsequently incubated with 10 ml HEK 293 lysate for 5 h under constant rotation at 4°C. Following incubation, the beads were washed three times with Tris-buffered saline. The beads were then washed once with phosphate-buffered saline (PBS). Co-immunoprecipitated RNA was extracted using phenol: chloroform: isoamyl alcohol (25:24:1; cat. no, 15593-031; Invitrogen; Thermo Fisher Scientific, Inc.). The RNA pellet was used for RT-qPCR analysis of GIT1 expression, using the aforementioned methods.

A BrdU assay was used to investigate the roles of miR-195 and GIT1 on cell proliferation. Briefly, the cultured cells (1×10⁵ chondrocytes) were seeded into 6-well plates and incubated for 24 h before miR-195 mimics and inhibitors, plasmids or siRNAs were transfected into cells using the Lipofectamine 2000 transfection reagent (Invitrogen; Thermo Fisher Scientific, Inc.). A BrdU Cell Proliferation Assay kit (Cell Signaling Technology, Inc.) was used to determine cell proliferation according to the manufacturer's instructions. Following transfection and incubation for 48 h, the medium was removed and cells were labeled with 10 mM BrdU for 3 h at 37°C. Cells were fixed and incubated with peroxidase-conjugated anti-BrdU antibody for 90 min at room temperature. Subsequently, the peroxidase substrate (3,3′,5,5′-tetramethylbenzidine) was added, and BrdU incorporation was quantitated by differences in absorbance at wavelength of 370 subtract wavelength of 492 nm. Cell proliferation was expressed as the mean percentage relative to the control values (set at 100%).

Cell migration rates were measured using a transwell chamber (BD Biosciences, Franklin Lakes, NJ, USA) containing Matrigel. The trypsinized chondrocytes were diluted to a final concentration of 2×10⁶ cells/ml in serum-free media, and 100 µl cell suspension was added into the upper chamber and 0.6 ml DMEM with 10% FBS was added into the lower chamber. Chambers were incubated at 5% CO₂ and 37°C for 6 h. Following removal of the medium, cells were fixed on the lower side of the insert filter by incubating with 4% paraformaldehyde (Sigma-Aldrich; Merck-Millipore) for 15 min, and cells that did not migrate on the upper side of the filter membrane were removed with a cotton swab. The cells on the lower side of the insert filter were stained with 0.1% Crystal Violet (Sigma-Aldrich; Merck-Millipore) for 10 min. The number of the cells on the lower side of the filter were then visualized and counted under a microscope (IX-70; Olympus Corporation, Tokyo, Japan) after washing with PBS (Gibco; Thermo Fisher Scientific, Inc.). The cells that had migrated through the membrane were stained and counted, and chondrocyte migration was expressed as the percentage of the total number of cells that had migrated.

Experiments were performed in triplicate and results are expressed as mean ± standard deviation. Statistical analyses were performed using the SPSS statistical software package (version, 17.0; SPSS, Inc., Chicago, IL, USA). Differences between control and treated groups were analyzed using non-parametric Mann-Whitney U tests. P<0.01 was considered to indicate a statistically significant difference.

Multiple miRNAs may target the same gene, and an miRNA can also target multiple genes, for example, miR-425, miR-376a and miR-138 may target GIT1, and miR-195 can target ZNF367, HDGF and CHEK1,. The present study used common bioinformatic algorithms to predict miRNAs that target GIT1. Previous studies have demonstrated that miR-195 serves a pivotal role in osteogenesis and bone development (17–19), however, there has been less research into the effect of other miRNAs on bone development, thus, the present study selected miR-195 for further investigation. As shown in Fig. 1A and B, the GIT1 3′-UTR was observed to contain miR-195 binding sites. In the present study, dual-luciferase reporter assay was used to verify binding of miR-195 to GIT1. As shown in Fig. 1B, luciferase expression levels decreased significantly following transfection of cells with vectors containing GIT1 3′-UTR clones plus miR-195 mimics (P<0.01). Conversely, luciferase expression levels were not significantly altered when the binding site was mutated (Fig. 1B). This demonstrated that miR-195 may bind to a site in the GIT1 3′-UTR, which suggests that GIT1 may be a target of miR-195.

Ago2 is a core component of the RNA-induced silencing complex that associates with miRNAs and their mRNA targets (20). Therefore, immunopurification of Ago2 under the appropriate conditions may retain associated miRNAs and mRNAs, thereby allowing miRNA targets to be identified. A monoclonal antibody against Ago2 was immobilized on Protein-G-Sepharose beads and incubated with HEK 293 cell lysates. Following stringent washing, the co-immunoprecipitated Ago-bound RNAs were extracted and subject to RT-qPCR analysis in order to detect GIT1 mRNA expression. Following transfection of miR-195 mimics, GIT1 mRNA levels were significantly higher when compared with controls (Fig. 1C; P<0.01). This further demonstrated that miR-195 may target and regulate GIT1 expression.

As GIT1 may be a target gene of miR-195, it is formally possible that miR-195 may regulate the expression of GIT1 in chondrocytes. In the current study, miR-195 mimics and inhibitors were transfected into human chondrocytes, and miR-195 and GIT1 expression was measured. The results indicated that a significant increase in miR-195 expression was associated with a significant downregulation in GIT1 mRNA and protein expression levels when compared with controls (Fig. 2; P<0.01). By contrast, when miR-195 expression was suppressed, GIT1 mRNA and protein expression increased significantly when compared with controls (Fig. 2B-D; P<0.01). These results suggest that miR-195 may regulate GIT1 expression in chondrocytes.

The results presented so far suggest that miR-195 targets and regulates the expression of GIT1 in chondrocytes. However, the role and association of this interaction with the biological behavior of chondrocytes requires further investigation. In the present study, a BrdU assay was performed in order to investigate the effect of miR-195 on chondrocyte proliferation. As shown in Fig. 3, chondrocyte proliferation increased significantly when miR-195 expression was suppressed with miR-195 inhibitors, as well as following overexpression of GIT1 compared with the control group (P<0.01). By contrast, transfection with miR-195 mimics or GIT1 siRNA demonstrated the opposite effect on chondrocyte proliferation compared with the control group (Fig. 3). These results demonstrate that chondrocyte proliferation may be inhibited by miR-195, but promoted by GIT1 expression. When miR-195 and GIT1 overexpression vectors were co-transfected, the inhibitory effect of miR-195 on chondrocyte proliferation was significantly attenuated (Fig. 3; P<0.01). However, upon co-transfection with miR-195 and GIT1 overexpression vectors containing the wild-type 3′-UTR sequence, miR-195-mediated inhibition of chondrocyte proliferation was unaffected (Fig. 3). These results demonstrate that miR-195 may be involved in mediating chondrocyte proliferation by regulating GIT1 expression.

Previous studies have demonstrated that a key function of GIT1 is to promote cell migration (21,22). Therefore, due to the observed putative role of miR-195 in regulating GIT1, it is possible that miR-195 may suppress cell migration. As shown in Fig. 4, upon transfection of miR-195 mimics in chondrocytes, the cell migration capacity decreased significantly when compared with controls (P<0.01). A similar effect was observed following transfection of cells with GIT1 siRNA (Fig. 4; P<0.01). Following transfection of miR-195 inhibitors, the migration capacity of chondrocytes increased significantly compared with the control group (P<0.01). This was similar to the migration capacity observed following transfection of cells with GIT1 expression vectors (Fig. 4; P<0.01). Co-transfection of miR-195 mimics with GIT1 expression vectors attenuated the inhibitory effect of miR-195 on cell migration (Fig. 4). These results suggest that miR-195 may inhibit chondrocyte migration by regulating GIT1 expression.