Human osteosarcoma cell lines (MG63 and U2OS) were obtained from The Cell Bank of Type Culture Collection of The Chinese Academy of Sciences and cultured in RPMI-1640 medium (Gibco; Thermo Fisher Scientific Inc.) with 10% FBS, 100 U/ml penicillin and 100 µg/ml streptomycin (Thermo Fisher Scientific Inc.). 293 T cells (Shanghai Gaining Biological Technology Co., Ltd.) were cultured in high-glucose DMEM medium with 10% FBS, which was used in luciferase reporter assay. All cells were cultured in an incubator under a water-saturated atmosphere of 5% CO₂−95% air at 37°C.

MG63 and U2OS cells (seeded in 12-well plates) were transfected with small interfering (si)-CCAT2 (Guangzhou RiboBio Co., Ltd.), anti-microRNA (miR)-143 oligodeoxyribonucleotide (AMO-143) (Ibsbio), or corresponding negative control (NC) sequences (scrambled sequences) 0.2 nmol/well at room temperature using the X-treme GENE siRNA transfection reagent according to the manufacturer's instructions (Roche Applied Science) and then cultured in an incubator in 5% CO₂−95% air at 37°C. Subsequent experimentation was performed within 48 h after transfection. The sequences were as follows. si-CCAT2, 5′-ACUCAUUGGUUCCUUUAAGGG-3′ and 5′-CUUAAAGGAACCAAUGAGUCC-3′; si-NC, 5′-ACAUCAUAGUCGAACUUUATT-3′ and 5′-GAAAAGGACACUAUGCGGCTT-3′; AMO-143, 5′-GCUACAGUGCUUCAUCUCAUU-3′ and AMO-NC, 5′-UACUCUUUCUAGGAGGUUGUGAUU-3′. Subsequent experimentation was performed within 48 h after transfection.

Proliferation of the MG63 and U2OS cells was evaluated using Cell Counting Kit-8 (CCK-8) (Dojindo Molecular Technologies Inc.). Cells were cultured for 2 h with the CCK-8 reagent prior to detection. The detailed procedure was described previously (12).

MG63 and U2OS cCells were inoculated into a 12-well culture plate (4×10⁵/well) in a monolayer. The cells were cultured with RPMI-1640 medium with 10% FBS (14,15). A straight line was then scratched on the monolayer surface (confluence of 70–80%) using a pipette tip (16). After treatment, the wells were washed twice to remove the dead cells from the medium. The width of the wound, which reflects the migration of the cells, was measured using a light microscope (Leica Microsystems GmbH) within 48 h.

MG63 and U2OS cells (1.5×10⁴/well) were inoculated into the upper chamber (RPMI-1640 medium without serum) of a 24-well Transwell plate (BD Biosiences), which was coated with BD Matrigel (BD Biosiences) for 5 h at 37°C. RPMI-1640 medium with 10% FBS was added to the lower chambers. Subsequently, 48 h after incubation at 37°C, invading cells in the lower chamber were stained with 0.1% crystal violet solution for 15 min at room temperature and the images was captured using a light microscope (Leica Microsystems GmbH).

Total RNA was extracted from MG63 and U2OS cells using TRIzol^® reagent (Roche Applied Science). cDNA was synthesized using the Takara reverse transcriptase kit (Takara Bio Inc.) according to the manufacturer's instructions. The reverse transcription steps were as follows: 3 cycles of 30°C for 10 min followed by 3 cycles of 50°C for 60 min followed by 95°C for 5 min and 4°C holding. The cDNA strand was amplified by RT-qPCR using SYBR-Green I (Toyobo Life Science) on a 7500 fast RT-qPCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The thermocycling conditions were as follows: 60 sec at 95°C followed by 40 cycles of 15 sec at 95°C, 15 sec at 60°C and 45 sec at 72°C. GAPDH and U6 were used as internal references for mRNA or miRNA, respectively. The primer sequences were as follows. CCAT2, forward 5′-CCCTGGTCAAATTGCTTAAC-3′ and reverse, 5′-TTATTCGTCCCTCTGTTTTATGG-3′; GAPDH, forward 5′-CCACATCGCTCAGACACC-3′ and reverse, 5′-ACCAGGCGCCCAATA-3′; miR-143, forward 5′-AGCGTGTGTCGTGGAGTC-3′ and reverse, 5′-TCGTGAGATGAAGCACTGTAG-3′; U6, forward 5′-CTCGCTTCGGCAGCACA-3′ and reverse, 5′-AACGCTTCACGAATTTGCGT-3′; and FOS-Like antigen 2 (FOSL2), forward 5′-GAGAGGAACAAGCTGGCTGC-3′ and reverse, 5′-GCTTCTCCTTCTCCTTCTGC-3′. The results were analyzed by the 2^−ΔΔCq method (17).

Total protein was exacted from MG63 and U2OS cells using RIPA Buffer (Beyotime Institute of Biotechnology) and quantified using a BCA kit (Beyotime Institute of Biotechnology). The proteins (20 µg per lane) were separated by 10% SDS-PAGE and transferred onto a nitrocellulose membrane. The nitrocellulose membrane was blocked with 5% skimmed milk for 2 h at room temperature. The membrane was incubated at 4°C with primary antibodies against FOSL2 (1:1,000; cat. no. PB0624; Wuhan Boster Biological Technology, Ltd.) and GAPDH (1:2,000; cat. no. TA890003; Origene Technologies Inc.) overnight, and incubated with anti-Rabbit IgG with ECM solution (1:5,000; cat. no. EK1002; Wuhan Boster Biological Technology, Ltd.) for 1 h at room temperature. The bands were visualized using ECM solution (Wuhan Boster Biological Technology, Ltd.). GAPDH was used as the internal loading control. The protein bands on the membrane were developed and quantified using Quantity One software v.4.6.2 (Bio-Rad Laboratories Inc.) (18).

lncRNA CCAT2 (gene name: CCAT2, Ensembl ID: ENSG00000280997) target was predicted using LncBase Predicted v.2 (http://carolina.imis.athena-innovation.gr/diana_tools/web/index.php?r=lncbasev2/index-predicted). The results indicated that miR-143 could bind to lncRNA CCAT2. 293T cells (2×10⁴/well) plated in a 24-well plate, were co-transfected with plasmids with wilt type CCAT2 (CCAT2-wt) or mutated CCAT2 (CCAT2-mut) (psiCHECKTM-2 Vector; Promega Corporation) (psiCHECK™-2 Vector; Promega Corporation) and miR-143 mimic or miR-NC (Ibsbio) by Lipofectamine^® 2000 (Thermo Fisher Scientific, Inc.). The sequences were as follows: miR-143, 5′-UGAGAUGAAGCACUGUAGCUC-3′ and miR-NC 5′-UCACAACCUCCUAGAAAGAGUAGA-3′. Luciferase activity was detected 24 h after transfection by the Dual-Luciferase Reporter Assay System (Promega Corporation). Renilla luciferase activity was normalized with CCAT2-wt + miR-NC group.

Data are presented as the mean ± standard deviation of at least 3 biological replicates. All data were analyzed using GraphPad Prism v.8 (GraphPad Software Inc.). The comparison of results among groups was performed by analysis of variance (ANOVA) and followed by the post hoc Tukey's multiple comparisons test. P<0.05 was considered to indicate a statistically significant difference.

LncRNA CCAT2 expression has been demonstrated to be higher in osteosarcoma tissues and cell lines compared with that in adjacent normal tissues and osteoblastic cell line, respectively (12,13). Compared with siNC group, lncRNA CCAT2 expression was downregulated after transfected with siRNA against CCAT2 in MG63 and U2OS cells (Fig. 1A and B). The proliferation of MG63 and U2OS cells was significantly inhibited by the downregulation of lncRNA CCAT2 compared with the siNC group (Fig. 1C and D).

Knockdown of lncRNA CCAT2 significantly inhibited the migration of MG63 and U2OS cells in the wound healing (Fig. 2A-D) and Boyden chamber cell invasion assays (Fig. 2E-H). The MG63 cell line had a more profoundly inhibited migration capability compared with U2OS cells (Fig. 2A-D).

LncBase Predicted v.2 software predicted that there were 3 binding sites between miR-143 and lncRNA CCAT2 (Fig. 3A). After transfection with miR-143 mimics, luciferase activity of 293T cells containing CCAT2-wild-type (CCAT2-wt) was lower compared with 293T cells containing the CCAT2-mutant (CCAT2-mut), which indicated that miR-143 could directly bind to lncRNA CCAT2 (Fig. 3B). In concert, miR-143 expression in MG63 and U2OS cells was significantly upregulated by downregulation of lncRNA CCAT2 (Fig. 3C and D).

Effects of lncRNA CCAT2 downregulation (si-CCAT2+AMO-NC group) on proliferation were attenuated by co-transfection with AMO-143 (si-CCAT2+AMO-143 group) in osteosarcoma cells (Fig. 4A and B). Similarly, the effects of lncRNA CCAT2 downregulation (si-CCAT2+AMO-NC group) on migration and invasion were attenuated by co-transfection with AMO-143 (si-CCAT2+AMO-143 group) in osteosarcoma cells (Fig. 4C-F).

FOSL2 is a target of miR-143 (19). The transfection of AMO-143 downregulated miR-143 expression compared with negative controls (Fig. 5A). Simultaneously, the expression of FOSL2 was upregulated after the interference of miR-143 compared with negative controls (Fig. 5B). Taken together, the FOSL2 mRNA and protein expressions were decreased by knockdown of lncRNA CCAT2 (Fig. 5C and D). However, co-transfection with AMO-143 reversed the role of lncRNA CCAT2 knockdown in regulating FOSL2 expression in the MG63 and U2OS cells (Fig. 5E and F).