The SaOS2, U2OS, MG63, OHS4 and CAL72 cell lines (American Type Culture Collection, Manassas, VA, USA) were cultured in Dulbecco's modified Eagle's medium (Invitrogen; Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal calf serum (FCS; Thermo Fisher Scientific) at 37°C in a humidified atmosphere containing 5% CO₂ in air.

TRIzol reagent (Invitrogen; Thermo Fisher Scientific) was used to isolate RNA according to the manufacturer's instructions, which was stored at −20°C. cDNA was synthesized using 3 µg RNA, which was denaturated and reverse-transcribed by using 300 U Moloney murine leukemia virus reverse transcriptase, 15 mg oligo dT primers and 1 mM deoxynucleoside triphosphate (dNTP) (Promega, Madison, WI, USA) in a total volume of 30 µl. SYBR Green Master Mix kit (ABGen, Courtaboeuf, France) was used for qPCR. A total of 0.5 mM of each primer (Invitrogen; Thermo Fisher Scientific) was used with sequences as follows: Human CTGF, forward 5′-CAG GCT AGA GAA GCA GAG CC-3′ and reverse 5′-GTA ATG GCA GGC ACA GGT CT-3′; β-actin, forward 5′-CTC CAT CCT GGC CTC GCT GT-3′ and reverse 5′-GCT GTC ACC TTC ACC GTT CC-3′. Thermocycling was conducted using the ABI 7500 (Applied Biosystems; Thermo Fisher Scientific) and the cycling conditions were as follows: Initial denaturation at 95°C for 15 min, followed by 40 cycles of 20 sec at 95°C, 15 sec at 58°C and 15 sec at 72°C, and final extension at 72°C for 7 min. The 2^−ΔΔCt method was used to determine the relative quantities of RNA.

In order to investigate the role of CTGF in OSA, cell lines were transduced with lentiviral vectors (LV) encoding either the full-length sequence (LV-CTGF) or a specific short hairpin (sh)RNA (sh-CTGF). The full-length CTGF ORF (1047 base pairs; GenBank accession number, CR541759.1) was amplified from the pFLAG-CMV2-CTGF plasmid (Invitrogen; Thermo Fisher Scientific). The primer sequences were as follows: Forward, 5′-TACTGGCGGCGGTATACCCG-3′ and reverse, 5′-TGCCATGTCTCCGTACAT-3′. The PCR product was inserted into the expression vector pcDNA3.1/myc-His(-)B-3X FLAG-IRES-hrGFP, derived from pcDNATM3.1/myc-His(-)B (Invitrogen; Thermo Fisher Scientific). Cell transduction was performed using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific) according to the manufacturer's instructions.

A bromodeoxyuridine (BrdU) incorporation assay was used to quantify cell replication. A previously described procedure was used in the present study (17). In brief, cells were cultured for 24 h in the presence of increasing concentrations of bisphosphonates (10⁻⁹–10⁻⁴ M) and labeled with BrdU for the last 6 h (kit purchased from GE Healthcare Life Sciences, Roosendaal, The Netherlands).

Double staining with ethidium bromide and acridine orange was performed to visualize and quantify the number of viable cells (green nuclei), apoptotic cells (nuclei condensed and colored orange), and necrotic cells (red nuclei). In briefly, 2 µl dye mixture (100 µg/ml acridine orange and 100 µg/ml ethidium bromide) was added to 20 µl cell suspension and immediately examined with the 40X oil immersion objective using a Leitz DMRB fluorescence microscope (green/red filter; 100 W lamp; Leica Microsystems GmbH, Wetzlar, Germany) equipped with a photometrics CCD camera and the Logikon image analysis system (Numeris Benelux SA, Ath, Belgium). Several fields, randomly chosen, were digitized and 600–800 nuclei for each sample were counted and scored. Results were expressed as the relative percentages of viable, apoptotic and necrotic cells to the total number of cells scored.

Effector caspase activity was performed as previously described (14,15). In brief, cells were treated with 10 mM atorvastatin (Adooq BioScience LLC, Irvine, CA, USA) or the solvent for 24 h then the caspase activity was determined. Cellular extracts (50 µg) were incubated with 0.2 mM acetyl-Asp-Glu-Val-Asp-p-nitroanilide (caspases-3, -6 and -7; Enzo Life Sciences, Inc., Farmingdale, NY, USA), Ac-LEHD-pNA (caspase-9; Enzo Life Sciences, Inc.) or Ac-IETD-pNA (caspase-8; Enzo Life Sciences, Inc.) as the substrates for the previously reported times (14,15) at 37°C in the presence or the absence of the specific caspase inhibitors Ac-DEVD-CHO, Ac-LEHD-CHO and Ac-IETD-CHO (10 µM). The specific activity (nmol of pNA/min/mg protein) was expressed as treated over control ratios.

A wound-healing assay was performed following the manufacturer's instructions (ibidi GmbH, Martinsried, Germany). A Transwell migration and invasion assay as performed as described previously (14). In brief, the cells (50,000 cells/insert) were incubated 2 h with or without statin and/or z-VAD-fmk prior to seeding into the inserts and incubation for a further 22 h. The cells that did not migrate through the filter were removed from the upper surface of the membrane using cotton-tipped swabs. The cells migrated to the underside were fixed in 3.7% paraformaldehyde in phosphate-buffered saline (PBS) at 4°C and stained with crystal violet (Amresco, Solon, OH, USA). The membranes were then cut from the insert and observed under a microscope (Axioplan 2 Imaging Mot Microscope System; Zeiss, Oberkochen, Germany). Five fields were randomly selected and counted and each assay was performed in duplicate.

A protocol of a previous study was used for the preparation of cell lysates (14) In brief, the proteins (30 µg) were resolved using 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE; Protogel, Atlanta, GA, USA) and transferred onto polyvinylidene difluoride nitrocellulose membranes (EMD Millipore, Billerica, MA, USA). The filters were incubated at room temperature for 2 h in 50 mm Tris-HCl (pH 7.4), 150 mm NaCl, 0.1% (v/v) Tween 20, 0.5% (w/v) bovine serum albumin (TBST/BSA) and then overnight at 4°C on a shaker with the rabbit monoclonal anti-GAPDH (ab181602) and anti-CTGF (ab6992) antibodies (1:1,000 in TBST/BSA; Abcam, Cambridge, UK). The membranes were washed twice with TBST and incubated for 2 h with the horseradish peroxidase-conjugated secondary antibody (1:20,000 in TBST/BSA). Following the final washes, the signals were visualized with Enhanced Chemiluminescence Western Blotting Detection Reagent (GE Healthcare Life Sciences) and autoradiographic film (X-Omat AR; Kodak, Rochester, NY, USA). Densitometric analysis using ImageQuant software was performed following digital scanning (Odyssey^® Fc; Agfa-Gevaert, Mortsel, Belgium).

A protocol of a previous study was used for the preparation of cell lysates (14,15). Incubation with rabbit monoclonal anti-GAPDH (ab181602; 1:200) and rabbit polyclonal anti-CTGF (ab6992; 1:200) antibodies was conducted at 4°C overnight. Cell extracts were collected in 2X loading lysis buffer [50 mM Tris-HCl (pH 6.8), 2% SDS, 10% 2-mercaptoethanol, 10% glycerol and protease inhibitor cocktail; Sigma-Aldrich, St. Louis, MO, USA]. The total cellular proteins were separated using 8% SDS–PAGE and transferred to Hybond-C nitrocellulose membranes (GE Healthcare Life Sciences, Chalfont, UK). Subsequent to blocking with PBS containing 5% BSA or nonfat milk, the membranes were incubated with the primary antibodies, followed by incubation with IRDye 800CW or 680RD secondary antibodies (1:10,000; LI-COR Biosciences, Lincoln, NE, USA). The protein bands were detected using the Odyssey Infrared Imaging System (Li-COR Biosciences).

Values are expressed as the mean ± standard deviation. Two-factor analysis of variance was used to compare values between groups, using SPSS software, version 19.0 (IBM SPSS, Armonk, NY, USA). P<0.05 was considered to indicate a statistically significant difference between values.

RT-qPCR analysis of CTGF was performed in the SaOS2, U2OS, CAL72, MG63 and OHS4 human OSA cell lines, revealing that CTGF mRNA was expressed in all cell lines, particularly in SaOS2 cells (Fig. 1A). Furthermore, the effect of statin treatment on the expression of CTGF was assessed in the OSA cell lines. CTGF mRNA expression in the panel of OSA cell lines was markedly decreased following treatment with statin (10 mM) (P<0.05 vs. untreated) (Fig. 1B). In addition, the effect of statin (10 mM) on the protein levels of CTGF in the panel of cell lines was assessed by immunoblot analysis, revealing that the protein levels of CTGF were decreased following statin (Fig. 1C). Collectively, these results indicated that statin treatment led to the downregulation of CTGF in human OSA cells. As the SaOS2 and U2OS cell lines expressed the highest and lowest levels of CTGF, respectively, they were selected to be used in the subsequent experiments.

A BrdU incorporation assay were used to determine the proliferative rates of transduced and parental cells, revealing that these were not affected by plasmid transduction (Fig. 2A). The results therefore indicated that CTGF had no significant effects on OSA-cell proliferation in human cell lines.

The present study investigated the effects of CTGF on OSA cell death. As shown in Fig. 2B, apoptotic and necrotic indices of sh-CTGF-transduced cells were higher than those of parental cells. By contrast, LV-CTGF-transduced cells displayed lower apoptotic and necrotic indices compared with those of parental cells. Furthermore, it was revealed that sh-CTGF-transduced cells exhibited increased caspase activity and an elevated Bax/Bcl2 ratio compared with those of parental cells. By contrast, caspase activity and the Bax/Bcl2 ratio were reduced in CTGF-overexpressing OSA cells compared with those in parental cells (Fig. 2C and D). These results indicated that CTGF expression was associated with the evasion of apoptosis by OSA cells.

The dose-dependent cytotoxic effects of doxorubicin, cisplatin and methotrexate on OSA cell viability are utilized for the chemotherapeutic treatment of OSA (14). The present study revealed that CTGF silencing significantly enhanced the caspase activity in SaOS2 cells following treatment with doxorubicin, cisplatin or methotrexate, whereas LV-CTGF slighly decreased caspase levels compared with those in native SaOS2 cells treated with the chemotherapeutics (Fig. 3A–C). It is therefore concluded that silencing of CTGF enhanced the efficacy of chemotherapeutic drugs against OSA.

The present study further investigated the invasiveness and migratory potential of transduced OSA cell lines, which represent the main characteristics of OSA progression and the development of metastasis. The results showed that CTGF silencing inhibited wound healing in sh-CTGF-transduced cells compared with that in parental cells, while CTGF overexpression enhanced wound healing (Fig. 4A). In addition, CTGF overexpression enhanced the migratory potential in a Transwell assay (Fig. 4B). The observed inhibition of the migratory potential by statin was not able to be rescued by overexpression of CTGF (Fig. 4C). Furthermore, a Transwell assay using Matrigel-coated inserts revealed that silencing of CTGF inhibited the invasive capacity of OSA cells, while cell invasiveness was promoted by CTGF overexpression (Fig. 4D). All of these results implied that CTGF had positive effects on cell migration and invasiveness in vitro, whereas invasion and migration were reduced in CTGF-silenced OSA cells. It can be concluded that CTGF expression is associated with the aggressiveness and metastatic potential of OSA cells.