Inhibiting proliferation and metastasis of osteosarcoma cells by downregulation of long non-coding RNA colon cancer-associated transcript 2 targeting microRNA-143.

Osteosarcoma is a malignant bone tumor, which has a high incidence in children and adolescents. However, the pathogenesis of osteosarcoma remains unclear. Long noncoding RNA (lncRNA) is a new potential therapeutic target and diagnostic biomarker for osteosarcoma. Hence, the present study aimed to explore the effect of lncRNA colon cancer-associated transcript (CCAT2) on osteosarcoma and its potential underlying mechanisms. For this purpose, the proliferation of osteosarcoma cells was measured using the CCK-8 assay. The scratch-wound and cell invasion assays were used to determine the migration and invasion of osteosarcoma cells, respectively. LncRNA CCAT2 and microRNA (miR)-143 binding sites were identified by the dual-luciferase reporter assay. RNA and protein expression levels were detected by reverse-transcription quantitative PCR and western blotting, respectively. Downregulation of lncRNA CCAT2 inhibited the proliferation, migration, and invasion of osteosarcoma cells. The findings also revealed that miR-143 bound directly to lncRNA CCAT2. The expression of miR-143 was upregulated by the knockdown of lncRNA CCAT2. Downregulation of the FOS-like antigen 2 was also observed after knockdown of lncRNA CCAT2. The function of lncRNA CCAT2 in osteosarcoma cells was attenuated by co-transfection with anti-miR-143 oligodeoxyribonucleotide. In conclusion, downregulation of lncRNA CCAT2 inhibited the proliferation and metastasis of osteosarcoma cells by targeting miR-143. lncRNA CCAT2 was identified as a potential target for osteosarcoma treatment. Copyright: © Bi et al.

Introduction

Osteosarcoma is a malignant bone tumor that accounts for 1–2% of all cancer cases 012-2016) (1). Although surgical and chemotherapy approaches for osteosarcoma have been greatly improved, the 5-year survival rate (2009–2015) was only 67% (birth to age 14) and ~ 69% (ages 15–19) (1). Hence, it is essential to determine novel and effective therapeutic targets and prognostic biomarkers for osteosarcoma.

Long non-coding RNAs (lncRNAs) are transcripts that are >200 nucleotides long (2). The regulatory RNA is abnormally expressed in various cancers, such as prostate cancer, liver cancer and lung cancer, making it an emerging diagnostic biomarker and therapeutic target (3–6). Numerous lncRNAs have been deregulated, and it has been proven that some are important regulators for osteosarcoma, including lncRNA cancer susceptibility candidate 2, maternally expressed gene 3 (MEG3) and colon cancer-associated transcript 2 (CCAT2) (7–9).

CCAT2 an oncogene, is highly expressed in cervical, colorectum, and lung cancer (9–11). The expression of CCAT2 is related to cancer progression and associated with poor overall survival in patients with cervical and colorectal cancer (10,11). CCAT2 is upregulated in non-small cell lung cancer tissues and cells, and promotes tumorigenesis by upregulating Pokemon expression (9). It has been revealed that the expression of lncRNA CCAT2 is higher in osteosarcoma tissues compared with that in adjacent normal tissues (12). High expression of lncRNA CCAT2 is related to larger tumor size, advanced stage, poor overall survival time and rate of patients with osteosarcoma (12). Similarly, Yan et al (13) also demonstrated that high expression of CCAT2 is associated with poor disease-free survival time and overall survival time in patients with osteosarcoma. In addition, lncRNA CCAT2 is upregulated in osteosarcoma cell lines (SOSP-9607, MG-63, U2OS, and SAOS-2) compared with a normal osteoblast cell line (hFOB) (13). Cell invasion is promoted by the overexpression of lncRNA CCAT2, and at least partially related to the upregulation of glycogen synthase kinase 3 β, large tumor suppressor 2 and c-Myc expression (12,13). However, the therapeutic potential and underlying mechanism regulating lncRNA CCAT2 in osteosarcoma remains elusive.

The present study was aimed to explore the therapeutic potential and molecular mechanism of CCAT2 in osteosarcoma. lncRNA CCAT2 may be a new potential target in the therapeutic of osteosarcoma.

Materials and methods

Cell culture

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.

Cell transfection

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.

CCK-8 assay

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).

Wound healing assay

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.

Boyden chamber cell invasion assay

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).

Reverse transcription-quantitative (RT-q) PCR

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).

Western blotting

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).

Luciferase reporter assay

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.

Statistical analysis

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.

Results

Downregulation of lncRNA CCAT2 inhibits the proliferation of osteosarcoma cells

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).

Figure 1.

Downregulation of lncRNA CCAT2 inhibits the proliferation of osteosarcoma cells. (A and B) Downregulated lncRNA CCAT2 expression in MG63 and U2OS cells transfected with siRNA targeting lncRNA CCAT2. Data was normalized to control group. (C and D) Reduced proliferation of MG63 and U2OS cells by downregulation of lncRNA CCAT2. **P<0.01, ***P<0.001 vs. si-NC. For panel A and B, n=3; for panel C and D, n=6. LncRNA; long-non-coding RNA; CCAT2, colon cancer-associated transcript 2; si, small interfering; NC, negative control; OD, optical density.

Downregulation of lncRNA CCAT2 hampers the migration capability and invasion of osteosarcoma cells

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).

Figure 2.

Downregulation of lncRNA CCAT2 inhibits the migration and invasion of osteosarcoma cells. (A and B) Representative scratch-wound assay images of MG63 and U2OS cells. Magnification, ×100. (C and D) Diminished migration capability of MG63 and U2OS cells due to downregulation of lncRNA CCAT2. (E and F) Representative Boyden chamber cell invasion assay images of MG63 and U2OS cells. Magnification, ×100. (G and H) Suppressed invasion capability of MG63 and U2OS cells due to downregulation of lncRNA CCAT2. *P<0.01, **P<0.001 vs. si-NC; n=3. LncRNA; long-non-coding RNA; CCAT2, colon cancer-associated transcript 2; si, small interfering; NC, negative control.

Downregulation of CCAT2 upregulates miR-143 expression in osteosarcoma cells

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).

Figure 3.

Downregulation of CCAT2 downregulates miR-143 expression in osteosarcoma cells. (A) Predicted binding sites between lncRNA CCAT2 and miR-143 using LncBase Predicted v.2. (B) Dual-luciferase reporter assay validated the direct binding site between miR-143 and lncRNA CCAT2 in 293T cells. (C and D) Downregulation of CCAT2-upregulated miR-143 expression in MG63 and U2OS cells. siRNA was used to knock down lncRNA CCAT2. &P<0.05 vs. CCAT2-wt+miR-NC; *P<0.05, **P<0.001 vs. si-NC; n=3. LncRNA; long-non-coding RNA; CCAT2, colon cancer-associated transcript 2; si, small interfering; NC, negative control; WT, wild-type; MUT, mutant; miR, microRNA.

Knockdown of miR-143 attenuates the effects of downregulation of lncRNA CCAT2 in osteosarcoma cells

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).

Figure 4.

Knockdown of miR-143 attenuates the effect of lncRNA CCAT2 downregulation on osteosarcoma cells. (A and B) Knockdown of miR-143 attenuated the effect of lncRNA CCAT2 downregulation on the proliferative capacity of MG63 and U2OS cells. Data was normalized to control group. (C) Representative scratch-wound assay images of MG63 and U2OS cells. Magnification, ×100. (D and E) Knockdown of miR-143 attenuated the effect of lncRNA CCAT2 downregulation on the migration capability of MG63 and U2OS cells. (F) Representative Boyden chamber cell invasion assay images of MG63 and U2OS cells. Magnification, ×100. (G and H) Knockdown of miR-143 attenuated the effect of lncRNA CCAT2 downregulation on the invasion capability of MG63 and U2OS cells. *P<0.05, ***P<0.001 vs. si-NC; #P<0.05, ##P<0.01, ###P<0.001 vs. si-CCAT2 + AMO-NC; For panel A and B, n=6; for panel D, E, G and H, n=3. LncRNA; long-non-coding RNA; CCAT2, colon cancer-associated transcript 2; si, small interfering; NC, negative control; miR, microRNA; AMO-143, anti-microRNA-143 oligodeoxyribonucleotide.

Downregulation of lncRNA CCAT2 inhibits FOSL2 expression by regulating miR-143 expression in osteosarcoma cells

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).

Figure 5.

Downregulation of lncRNA CCAT2 inhibits FOSL2 expression through the regulation of miR-143 in osteosarcoma cells. (A and B) AMO-143 transfection-induced downregulated miR-143 expression and upregulated FOSL2 mRNA expression. (C and D) Downregulation of lncRNA CCAT2 inhibited FOSL2 mRNA expression through miR-143 upregulation in MG63 and U2OS cells. (E and F) Downregulation of lncRNA CCAT2 inhibited FOSL2 protein expression through miR-143 upregulation in MG63 and U2OS cells. *P< 0.05, **P<0.01, ***P<0.001 vs. si-NC; #P<0.05 vs. si-CCAT2 + AMO-NC; n=3. LncRNA; long-non-coding RNA; CCAT2, colon cancer-associated transcript 2; si, small interfering; NC, negative control; miR, microRNA; AMO-143, anti-microRNA-143 oligodeoxyribonucleotide; FOSL2, FOS-like antigen 2.

Discussion

Osteosarcoma is a malignant solid tumor with high incidence in children and adolescents (20,21). In the present study, the role and molecular mechanism of lncRNA CCAT2 in osteosarcoma cells was investigated and the results indicated that downregulation of lncRNA CCAT2 expression regulated miR-143 expression and inhibited the proliferation and metastasis of osteosarcoma cells.

Several studies have demonstrated that lncRNAs are major regulators of gene expression and essential in a variety of pathological procedures, including cancer, cardiovascular diseases and neuronal disorders (22–25). In addition, the expression profiles of lncRNAs are altered in cancer (26,27). The role and therapeutic potential of cancer-related lncRNAs have become a research hotspot in recent years. Although a limited number of lncRNAs have been studied, researchers have made significant progress in studying the specific mechanism of lncRNA regulation. Recent studies have reported the role of lncRNAs in the generation and progression of cancers and how they can be used as biomarkers for diagnosis and treatment of cancers (28–30).

CCAT2 is a new oncogenic lncRNA gene, which was first discovered in related studies on colon cancer (31). It was overexpressed in microsatellite-stable colorectal cancer to promote proliferation, metastasis, and chromosomal instability of the tumor (31). The effect of lncRNA CCAT2 has been demonstrated in different types of cancers. For example, lncRNA CCAT2 has been reported to be upregulated in lung cancer, which not only promotes the generation and progression of lung cancer, but is also considered to be a predictive factor for prognosis (9,32). LncRNA CCAT2 expression is related to poor prognosis in patients with gastric cancer (33,34). The expression of lncRNA CCAT2 is higher in hepatocellular carcinoma cell lines compared with that in normal liver epithelial cells (35). In addition, the high expression of CCAT2 significantly suppressed hepatocellular carcinoma cell apoptosis (35–37).

LncRNA CCAT2 expression is significantly higher in osteosarcoma tissues and cells compared with that in normal tissues and cells (12,13). In the present study, to investigate the role of lncRNA CCAT2 in osteosarcoma cells, the expression of lncRNA CCAT2 was downregulated using siRNA. This resulted in the inhibition of proliferation and migration of osteosarcoma cells in the present study. LncRNA CCAT2, a competitive endogenous RNA can regulate gene expression by sponging miRNAs (38). In the present study, an online software, LncBase Predicted v.2, predicted that lncRNA CCAT2 has three miR-143 binding sites. Subsequently, a direct interaction between lncRNA CCAT2 and miR-143 was revealed in the dual-luciferase reporter assay. In concert, in the present study miR-143 expression was significantly upregulated by the downregulation of lncRNA CCAT2.

miR-143 is a tumor-suppressor miRNA in colon cancer, breast cancer and pancreatic cancer, etc (39–41). In addition, the proliferation and migration of osteosarcoma cells have been demonstrated to be inhibited by the upregulation of miR-143 (42,43). In the present study, to confirm the effect of miR-143 regulation on lncRNA CCAT2, miR-143 expression was downregulated using AMO-143. The findings of the present study revealed that downregulation of lncRNA CCAT2 inhibited tumor proliferation and metastasis by regulating miR-143.

Recent studies have suggested that miR-143 expression is decreased in osteosarcoma tissues and cell lines (MG-63 and 143B) compared with that in adjacent normal tissues and osteoblastic cell line (hFOB 1.19), respectively (19,44). In addition, FOSL2, also known as Fra-2 is associated with metastasis in breast and lung cancer (42,43). Tumor proliferation and migration was inhibited by upregulation of miR-143 targeting FOSL2 (19). In accordance with this, the present study also demonstrated that the inhibition of miR-143 could upregulate FOSL2 mRNA expression in osteosarcoma cells.

Subsequently, in the present study the effect of regulation of lncRNA CCAT2 on FOSL2 expression was studied and it was revealed that FOSL2 expression was inhibited by the downregulation of lncRNA CCAT2, whereas co-transfection with AMO-143 reversed the effect of lncRNA CCAT2 on FOSL2. Hence, the downregulation of lncRNA CCAT2 inhibited FOSL2 expression through the upregulation of miR-143.

In conclusion, the present study improved understanding of the pathogenesis of osteosarcoma. In addition, the findings of the present study indicated that lncRNA CCAT2 may be a new potential therapeutic target for osteosarcoma. An improved understanding of the importance of deregulated lncRNAs will definitely improve osteosarcoma prevention and treatment.