Antibodies

For immunoblotting, anti-CTGF (CCN2) (Abcam, Cambridge, UK), β-actin (Fujifilm, Tokyo, Japan), anti-Aggrecan (Cosmo Bio Co, Tokyo, Japan), anti-SOX9 (Merck, Darmstadt, Germany), and anti-TypeII collagen (Proteintech, Rosemont, IL, USA), antibodies were employed. The secondary antibodies, horseradish peroxidase (HRP)-conjugated anti-rabbit IgG and anti-mouse IgG were purchased from Cell Signaling Technology (Danvers, MA, USA).

Cell culture

Human chondrocytic cell line HCS-2/8 cells that stably retain mature chondrocytic phenotype and murine chondrocyte progenitor cell line ATDC5 cells were cultured in Dulbecco′s Modified Eagle’s Medium (DMEM) and alpha-modified Eagle’s minimum essential medium (αMEM) containing 10% fetal bovine serum (FBS), respectively. All cells were cultured at 37 °C in humidified air with 5% CO2. To induce ATDC5 cell differentiation, the culture medium was replaced with fresh αMEM containing 5% FBS and ITS (insulin–transferrin–selenium; Corning, Corning, NY, USA) every 3 days after the cells reached confluence.

RNA extraction

HCS-2/8 cell RNA was harvested 3 days after cell seeding. ATDC5 cell RNA was harvested every 3 days from 0 to 21 days after differentiation induction. RNA extraction from HCS-2/8 and ATDC5 cells was performed with the RNeasy® Mini Kit (Qiagen, Hilden, Germany) according to the protocol provided by the manufacturer.

Ribonuclease R treatment

To remove linear RNA from the extracted RNA, 1 μg of RNA was incubated with 0 U, 1 U, 5 U, and 10 U of Ribonuclease R (RNaseR; AR BROWN, Tokyo, Japan) in the attached reaction buffer (0.2 M Tris–HCl, 1 M KCl, and 1 mM MgCl2) at 37 °C for 15 min, followed by an inactivation process at 95 °C for 3 min.

Reverse transcription (RT) polymerase chain reaction (PCR)

Five hundred nanograms of RNA was reverse-transcribed with PrimeScript cDNA synthesis kits (Takara, Japan). Thereafter, PCR was performed according to the protocol of Quick Taq HS DyeMix (Toyobo, Osaka, Japan). The number of PCR cycles was within the range of 30–40 cycles in each experiment. For circRNA detection, primer sets were designed to specifically amplify the segments containing the back-splice junctions of human and murine CCN2-derived circRNAs (Table 1).

Table 1 Primers for amplifying CCN2- and Ccn2-drived circRNAsAgarose gel electrophoresis

PCR products were separated via electrophoresis on 1.5% or 2% agarose gels with Tris–acetate–EDTA buffer containing ethidium bromide and visualized with a UV transilluminator (Kurabo, Osaka, Japan). The intensities of the visualized bands were quantified using the image processing software ImageJ (https://imagej.nih.gov/ij/).

DNA sequencing

The bands containing the target DNA fragments were excised from the agarose gels. The DNA was purified according to the protocol of the QlAquick Gel Extraction Kit (Qiagen). The purified DNA was ligated into pGEM®-T Easy Vector (Promega, Madison, WI, USA) using the attached ligase mixture. Thereafter, plasmid DNA amplified with Escherichia coli was extracted using the GenEluteTM Plasmid Miniprep Kit (Merck). The nucleotide sequences of the plasmid DNA from the Sp6 and T7 primer binding sites were determined using a commercial service (FASMAC, Atsugi, Japan). The obtained nucleotide sequences were analyzed with the online tools and databases provided by the National Center for Biotechnology Information (NCBI: https://www.ncbi.nlm.nih.gov).

RNAi

A 27-mer siRNA duplex containing 2 deoxynucleotides (5′-AAU AUU GUG UGU GUG ACG AGC CC dAdA-3′and 5′- UUG GGC UCG UCA CAC ACA CAA UAU UUA-3′) was designed for the specific knockdown of the CCN2-derived circRNA (A), targeting the back-splice junction. This siRNA duplex and a negative control siRNA were obtained from Nippon Gene (Tokyo, Japan). The siRNA duplex was transfected into HCS-2/8 cells (400,000 cells per well in 6-well plates) using siPORT™ NeoFX™ Transfection Agent (Thermo Fisher Scientific, Waltham, MA, USA) and incubated at 37 °C in humidified air with 5% CO2. After 24 h, the medium was replaced with a fresh one, and the cells were incubated for another 24 h. Thereafter, the RNAs were collected for subsequent analyses.

Quantitative real-time PCR (qPCR)

The real-time quantitative monitoring of PCR amplification was performed using the SYBR Green Realtime PCR Master Mix (Toyobo) according to the manufacturer’s protocol. The fluorescent signals were detected and analyzed using the StepOnePlus™ Real-Time PCR System (Thermo Fisher Scientific). The primers used for the qPCR analysis are described in Table 2.

Table 2 Primers for real-time PCRImmunoblotting

HCS-2/8 cells were lysed in a RIPA buffer (50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 1% Igepal CA-630, 0.5% deoxycholate, and 0.1% sodium dodecyl sulfate: SDS). Aggrecan was processed by chondroitinase ABC 0.1 unit/mg CS (Merck, Darmstadt, Germany) in 50 mM Tris acetate, 10 mM EDTA, pH 7.6 for 1 h at 37 °C, followed by boiling at 95 °C for 5 min in 0.5% SDS, 20 mM dithiothreitol (Lark et al. 1995). The lysate diluted in 1 × SDS sample buffer (50 mM Tris–HCl, pH6.8, 2% SDS, 5% glycerol, 2% bromophenol blue, 5% 2-mercaptoethanol) was boiled at 95 °C for 3 min. The lysate was subjected to SDS–polyacrylamide gel electrophoresis in 6%, 8%, and 10% polyacrylamide gels. Proteins were transferred onto polyvinylidene difluoride membranes with a wet or semi-dry blotting apparatus. The membranes were then incubated for 1 h in a blocking buffer [5% non-fat milk in phosphate-buffered saline (PBS)] and subsequently incubated overnight at 4 °C with anti-CCN2 (1:1000), β-actin (1:1000), anti-Aggrecan (1:10), anti-SOX9 (1:500), and anti-TypeII collagen (1:400) antibodies in the blocking buffer. The membranes were incubated with HRP-conjugated anti-rabbit IgG (1:2000) and anti-mouse IgG (1:2000) antibodies in the blocking buffer at room temperature for 1 h.

Glycosaminoglycans (GAG) assay

After 72 h of HCS-2/8 cell culture, the culture supernatants were collected. Cellular proteins were extracted by 2%Triton X-100/PBS. The lysate was treated at 65 °C for 90 min with 1 mg/ml actinase E in 0.2 M Tris–HCl (pH 7.5) and 5 mM CaCl2. Twenty microliters of the supernatant or cell lysate were added to 200 μl of 1,9 dimethylmethlyene blue (DMMB) reagent (38.46 μM DMMB, 40.5 mM glycine, 27.38 mM NaCl, 0.6% acetic acid). Thereafter, the absorbance was read using a plate reader at a wavelength of 525 nm immediately. GAG amount was normalized to that of the total protein. The total protein amount was measured with the BCA protein assay kit (Thermo Fisher Scientific) according to the manufacturer’s protocol.

Luciferase reporter assay

The miR-181a reporter construct pGL3-181TS with a miR-181-5p target sequence in the CCN1 mRNA 3′-UTR was produced in a previous study (Sumiyoshi et al. 2013) by using pGL3-L(+) vector containing a firefly luciferase gene (Kubota et al. 1999). HCS-2/8 cells were seeded in 12-well plates at approximately 2 × 105 cells per well. Seven hundred nanograms of pGL3-181TS and 350 ng of phRL-TK (int-) (Promega, Madison, WI, USA) transfection was performed with X-tremeGENE 9 DNA transfection reagent (Merck) into HCS-2/8 cells transfected with si-circRNA (A) or control siRNA. After 48 h, luciferase activity was measured using the Dual-Luciferase Reporter Assay Kit (Promega). Firefly luciferase activity was normalized to Renilla luciferase activity.

RNA analysis in silico

The secondary structure prediction of human CCN2 pre-mRNA was performed online with UNAfold (http://www.unafold.org). Putative miRNA binding sites were searched in silico on the TargetScan website (https://www.targetscan.org/vert_80/).

Statistical analysis

The results are presented as mean ± standard deviation. The statistical significance of the differences between the mean values was determined with Dunnett’s test or Student’s t-test. Differences between the mean values were considered significant at a P-value of < 0.05.