LncRNA TTN-AS1 regulates osteosarcoma cell apoptosis and drug resistance via the miR-134-5p/MBTD1 axis

LncRNA TTN-AS1 is highly expressed in osteosarcoma and is associated with poor prognosis

Bioinformatics analysis revealed that lncRNA TTN-AS1 was upregulated in osteosarcoma (Figure 1A). Clinical sample test results also showed that lncRNA TTN-AS1 was highly expressed in tumour tissues (2.08 ± 2.45 vs. 1.00 ± 0.02) (Figure 1B). Overexpression of lncRNA TTN-AS1 was also present in osteosarcoma cell lines (5.09 ± 0.94 and 3.47 ± 1.04 vs. 1.00 ± 0.14) (Figure 1C). Patients with high expression of lncRNA TTN-AS1 had lower survival rates (Figure 1D). Further clinical analysis also showed that a high level of lncRNA TTN-AS1 was associated with higher Enneking stage and remote transfer (Table 1).

Figure 1. lncRNA TTN-AS1 is highly expressed in osteosarcoma and is associated with poor prognosis. (A) Bioinformatics analysis of differentially expressed genes in osteosarcoma. (B) QPCR was used to detect the expression level of lncRNA TTN-AS1 in tumour tissues and adjacent tissues. (C) QPCR was applied to detect lncRNA TTN-AS1 levels in osteosarcoma cell lines. (D) 5-year survival analysis was done on osteosarcoma patients with different lncRNA TTN-AS1 levels. ^*P < 0.05 vs. normal tissue or hFOB 1.19 cell group;

LncRNA TTN-AS1 promotes cell viability and inhibits apoptosis

QPCR results showed that the TTN-AS1 level in the sh-TTN-AS1-1 and sh-TTN-AS1-2 groups was lower than that in the sh-NC group (Saos-2: 0.24 ± 0.08 and 0.55 ± 0.17 vs. 1.00 ± 0.19; U-2OS: 0.25 ± 0.04 and 0.54 ± 0.23 vs. 1.00 ± 0.10), while vector-TTN-AS1 significantly upregulated the level of TTN-AS1 (Saos-2: 4.06 ± 0.92 vs. 1.00 ± 0.09; U-2OS: 4.25 ± 0.89 vs. 1.00 ± 0.06), indicating successful transfection (Figure 2A–2D). Sh-TTN-AS1-1 was used for subsequent experiments and referred to as sh-TTN-AS1. The relative cell viability in the vector-TTN-AS1 group (Saos-2: 7.24 ± 0.35; U-2OS: 7.60 ± 0.47) was significantly higher than that of the vector-NC group (Saos-2: 5.28 ± 0.31; U-2OS: 5.88 ± 0.28). In addition, the relative cell viability in the sh-TTN-AS1 group (Saos-2: 4.08 ± 0.11; U-2OS: 4.01 ± 0.15) was lower than that of the sh-NC group (Saos-2: 5.02 ± 0.13; U-2OS: 1.13 ± 0.14) (Figure 2E–2F). The apoptosis rate was increased after transfection with sh-TTN-AS1 (Saos-2: 11.04 ± 2.44%; U-2OS: 10.49 ± 0.20%), and the apoptosis rate was decreased after transfection with vector-TTN-AS1 (Saos-2: 3.92 ± 0.08%; U-2OS: 3.62 ± 0.04%) (Figure 2G–2H). This indicated that lncRNA TTN-AS1 promoted cell viability and inhibited apoptosis.

Figure 2. lncRNA TTN-AS1 promoted cell viability and inhibited apoptosis. (A–D) QPCR was used to detect the expression level of lncRNA TTN-AS1. (E, F) The cell viability of each group of cells was detected by the CCK-8 method. (G, H) The apoptosis rate of each group of cells was tested using the CCK-8 method. ^*P < 0.05 vs. sh-NC group; ^#P < 0.05 vs. vector-NC.

LncRNA TTN-AS1 targets miR-134-5p

The predicted results from the bioinformatics analysis showed that lncRNA TTN-AS1 targeted miR-134-5p (Figure 3A). Clinical sample test results showed that miR-134-5p was expressed at low levels in osteosarcoma (0.52 ± 0.41) (Figure 3B). Further experimental results showed that upregulation of lncRNA TTN-AS1 inhibited the expression of miR-134-5p, and downregulation of lncRNA TTN-AS1 gave the opposite results (Figure 3C–3D). Promotion or inhibition of miR-134-5p expression had no significant effects on lncRNA TTN-AS1 (Figure 3E–3F). Furthermore, the mutated lncRNA TTN-AS1 did not cause changes in the level of miR-134-5p (Figure 3G–3H). This indicated that lncRNA TTN-AS1 could be a target to regulate the level of miR-134-5p.

Figure 3. lncRNA TTN-AS1 targeted miR-134-5p. (A) The website predicted that lncRNA TTN-AS1 targeted miR-134-5p. (B) QPCR was used to detect the expression level of miR-134-5p in tumour tissues and adjacent tissues. (C, D) The effects of downregulation or upregulation of lncRNA TTN-AS1 on miR-134-5p are shown. (E, F) The effects of downregulation or upregulation of miR-134-5p on lncRNA TTN-AS1 are shown. (G, H) The effects of transfected mutant lncRNA TTN-AS1 on miR-134-5p are shown. ^*P < 0.05 vs. normal tissue or vector-NC.

MiR-134-5p targets MBTD1, which is regulated by lncRNA TTN-AS1

The TargetScan website was used to predict the target gene of miR-134-5p (Figure 4A). The luciferase assay demonstrated that miR-134-5p targeted the 3′-UTR region of MBTD1 (Figure 4B, 4C). Further studies also showed that compared with the inhibitor-NC group, inhibition of miR-134-5p levels could upregulate MBTD1 mRNA and protein levels (Saos-2: 3.14 ± 0.67 and 1.98 ± 0.55; U-2OS: 2.21 ± 0.13 and 2.07 ± 0.57) (Figure 4D–4H). This indicated that miR-134-5p can directly target MBTD1.

Figure 4. MiR-134-5p targets MBTD1, which is regulated by lncRNA TTN-AS1. (A) The website predicted that MBTD1 was the target gene of miR-134-5p. (B, C) The luciferase assay confirmed that miR-134-5p targets MBTD1. (D–H) The effects of down-regulating miR-134-5p on MBTD1 mRNA and protein expression levels are shown. (I–K) The effects of upregulation of lncRNA TTN-AS1 on MBTD1 protein expression are shown. (L–N) The effects of knockdown of lncRNA TTN-AS1 and miR-134-5p on MBTD1 protein expression are shown. ^*P < 0.05 vs. mimic-NC, inhibitor-NC or sh-NC + inhibitor-NC group; ^#P < 0.05 vs. sh-TTN-AS1 + inhibitor-NC.

The results also showed that upregulation of lncRNA TTN-AS1 promoted MBTD1 protein expression (Saos-2: 2.27 ± 0.44; U-2OS: 1.98 ± 0.48) (Figure 4I–4K). In addition, the results showed that downregulation of lncRNA TTN-AS1 levels inhibited MBTD1 protein expression (Saos-2: 0.26 ± 0.03; U-2OS: 0.38 ± 0.03), while inhibitors partially reversed the inhibitory effects of lncRNA TTN-AS1 on MBTD1 (Saos-2: 0.82 ± 0.41; U-2OS: 1.17 ± 0.42) (Figure 4L–4N). This suggested that lncRNA TTN-AS1 could regulate the expression level of MBTD1 by targeting miR-134-5p.

MBTD1 is highly expressed in osteosarcoma and is associated with poor prognosis, and MBTD1 promotes cell viability and inhibits apoptosis

The level of MBTD1 in cancer tissues was significantly higher than that in normal tissues (Figure 5A, 5B). Osteosarcoma patients with high expression of MBTD1 had worse 5-year survival rates compared to those with low expression of MBTD1 (50.005 vs. 26.43%) (Figure 5C). Western blotting results showed that MBTD1 protein levels in the sh-MBTD1-1 and sh-MBTD1-2 groups were significantly lower than those in the sh-NC group, indicating successful transfection experiments (Figure 5D–5E). sh-MBTD1-1 was used for subsequent experiments and referred to as sh-MBTD1. Cell lines overexpressing MBTD1 were also successfully established, and MBTD1 protein levels were upregulated 3.85 (± 0.38) and 3.67 (± 0.72) times in Saos-2 and U-2OS, respectively (Figure 5G–5H). Further studies showed that overexpression of MBTD1 promoted cell viability and inhibited apoptosis, while downregulation of MBTD1 inhibited cell viability and promoted apoptosis (Figure 5G–5L). This suggested that MBTD1 had a role in promoting tumour growth.

Figure 5. MBTD1 is highly expressed in osteosarcoma and associated with poor prognosis, and MBTD1 promotes cell viability and inhibits apoptosis. (A, B) The expression level of MBTD1 in osteosarcoma tissues and adjacent tissues is shown. © 5-year survival analysis of osteosarcoma patients with different MBTD1 levels is shown. (D–H) The effects of MBTD1 knockdown or overexpression on MBTD1 protein expression levels are shown. (I–L) The effect of knockdown or overexpression of MBTD1 on cell viability and apoptosis is shown. ^*P < 0.05 vs. normal tissue or sh-NC group; ^#P < 0.05 vs. vector-NC.

LncRNA TTN-AS1 inhibited tumour growth via MBTD1.

The relative cell viability of the vector-TTN-AS1 + sh-NC group (Saos-2: 7.95 ± 0.27; U-2OS: 7.39 ± 0.66) was significantly higher than that of the vector-NC group; the cell viability of the Vector-TTN-AS1 + sh-MBTD1 group (Saos-2: 4.92 ± 0.18; U-2OS: 4.20 ± 0.31) was lower than that of the Vector-TTN-AS1 + sh-NC group (Figure 6A–6B). The relative apoptosis rate of the Vector-TTN-AS1 + sh-NC group (Saos-2: 3.17 ± 0.62; U-2OS: 3.72 ± 0.48) was significantly lower than that of the vector-NC group; the cell viability of the Vector-TTN-AS1 + sh-MBTD1 group (Saos-2: 7.11 ± 0.64; U-2OS: 6.08 ± 0.40) was higher than that of the Vector-TTN-AS1 + sh-NC group (Figure 6C–6D). These results suggested that knockdown of MBTD1 reversed the cancer-promoting effects of overexpression of lncRNA TTN-AS1. Animal experiments showed that the tumour weight of the vector-TTN-AS1 + sh-NC group (Saos-2: 2.66 ± 0.69; U-2OS: 2.24 ± 0.43) was significantly higher than that of the vector-NC group; the cell viability of the vector-TTN-AS1 + sh-MBTD1 group (Saos-2: 0.99 ± 0.18; U-2OS: 0.76 ± 0.11) was lower than that of the Vector-TTN-AS1 + sh-NC group (Figure 6E–6F).

Figure 6. lncRNA TTN-AS1 inhibits tumour growth via MBTD1. (A–D) The effects of overexpression of lncRNA TTN-AS1 and knockdown of MBTD1 on cell viability and apoptosis are shown. (E, F) The effects of overexpression of lncRNA TTN-AS1 and knockdown of MBTD1 on tumour growth are shown. ^*P < 0.05 vs. vector-NC + sh-NC group; ^#P < 0.05 vs. vector-TTN-AS1 + sh-NC.

Downregulation of lncRNA TTN-AS1 reduces drug resistance

The cell viability of the osteosarcoma cell lines Saos-2 and U2OS treated with different concentrations of cisplatin was examined. The cell viability decreased by approximately 50% when the concentration of cisplatin was 10 μM (Figure 7A–7B). The relative cell viability of the sh-TTN-AS1 group (Saos-2: 0.27 ± 0.02; U-2OS: 0.15 ± 0.01) was significantly lower than that of the sh-NC group (Saos-2: 0.46 ± 0.04; U-2OS: 0.45 ± 0.05) in the presence of 10 μM cisplatin (Figure 7C–7D). The relative cell viability of the vector-TTN-AS1 group (Saos-2: 0.96 ± 0.47; U-2OS: 0.78 ± 0.30) was significantly higher than that of the sh-NC group (Saos-2: 0.75 ± 0.02; U-2OS: 0.51 ± 0.03) in the presence of 10 μM cisplatin (Figure 7E–7F). Upregulation of lncRNA TTN-AS1 partially reverses the inhibitory effect of cisplatin on cells.

Figure 7. Downregulation of lncRNA TTN-AS1 reduces drug resistance. (A, B) The effects of different concentrations of cisplatin on the viability of osteosarcoma cells are shown. (C, D) Downregulation of lncRNA TTN-AS1 enhanced the inhibitory effects of cisplatin on osteosarcoma cells. (E, F) Upregulation of lncRNA TTN-AS1 attenuated the inhibitory effect of cisplatin on osteosarcoma cells. ^*P < 0.05 vs. sh-NC and vector-NC group of 0 μM; ^#P < 0.05 vs. sh-NC and vector-NC group of 10 μM.

Bioinformatics analysis

The osteosarcoma patient gene data (GSE12865) were downloaded from the GEO database (https://www.ncbi.nlm.nih.gov/geo/). A difference analysis was performed on the data. The miRcode website (http://www.mircode.org/) performed a biosignal analysis to predict miRNAs downstream of lncRNA TTN-AS1. The TargetScan website (http://www.targetscan.org/vert_72/) was used to predict downstream target proteins of miR-134-5p.

Osteosarcoma tissues and research

55 osteosarcoma tissues and 9 adjacent tissues were purchased from Alenabio Biotechnology Co., Ltd. (OS208a, Xi’an, China). Quantitative polymerase chain reaction (qPCR) was used to detect lncRNA TTN-AS1, miR-134-5p and MBTD1 mRNA expression levels in cancer tissues and normal tissues. The 5-year survival of patients with high or low expression of lncRNA TTN-AS1 and MBTD1 mRNA was compared. The correlation between lncRNA TTN-AS1 and pathological features of patients with osteosarcoma was analysed. Approval was obtained from the Tongren Hospital Ethics Committee.

Cells culture and transfection

Human osteoblast hFOB 1.19 and osteosarcoma cell lines Saos-2 and U-2OS were purchased from American Type Culture Collection (Manassas, VA, USA) and were grown in RPMI-1640 medium containing 10% foetal bovine serum (FBS, Thermo Fisher, Waltham, USA), 50 U/mL penicillin and 50 μg/mL streptomycin (15070063, Thermo Fisher, Waltham, USA). The plasmid constructs and the corresponding negative control (NC) were built by Shanghai Genepharma Company (China). Plasmid transfection technology was applied to silence or overexpress lncRNA TTN-AS1, miR-134-5p and MBTD1. The cells were transfected with 100 nM RNA oligonucleotides using LipofectamineTM 2000 (Invitrogen, Waltham, USA). Cells were harvested 48 h after transfection for subsequent experiments.

QPCR

Total RNA was acquired using TRIzol (Invitrogen, USA). The cDNA was generated from 1 μg isolated miRNA/mRNA by using an iScriptTMcDNA Synthesis Kit (Bio-Rad, USA). For qPCR processing, the FastStart Universal SYBR Green Master kit (Roche, Switzerland) was used. The reaction system was prepared as follows: 2.5 μL cDNA template, 1 μL forward primer (10 μM), 1 μL reverse primer (10 μM), 10 μL 2 × SYBR Green master mix, 5.5 μL dH₂O. QPCR was performed using the following procedure: 2 min at 95°C, 40 cycles of 15 sec at 95°C, 25 sec at 60°C and 60 sec at 72°C. U6 acted as an internal reference for miR-134-5p, and GAPDH served as an internal reference for lncRNA and mRNA. The primers were synthesized by Sangon Biotech (Shanghai, China) and are shown in Table 2.

Western blot analysis

The whole-cell protein was extracted by using ice-cold RIPA buffer mixed with protease inhibitors. The protein lysate was separated by SDS-PAGE at 110 V for 100 min and transferred to PVDF membranes at 90 V for 90 min. The PVDF membrane was blocked in 5% non-fat milk for 1 h at room temperature, and antibody (MBTD1, ab170848, Abcam, USA, 71 kD; GAPDH, ab9484, 36 kD) was added at 4°C overnight. The membrane was further probed with the secondary antibody IgG H&L (HRP) (ab6721, Abcam, San Francisco, USA, 1:5000) after washing the membrane with PBST (PBS with 0.2% Tween 20). The protein bands were detected by Pierce ECL Plus western blotting substrate (Thermo Fisher, Waltham, USA) on a ChemiDoc MP instrument (Bio-Rad, California, USA).

Cell counting kit 8 (CCK-8) assay

The cells were seeded in a 96-well culture plate (2 × 10⁴ cells / mL, 100 μL / well), and the blank controls (with medium alone) were set. After hypoxia-reoxygenation, 10 μL of CCK-8 (Beyotime Institute of Biotechnology, Beijing, China) reagent was added to each well, and the culture was continued for 2 h at 37°C. The absorbance values were measured at 450 nm on a microplate reader (Tecan Infinite M200 Micro Plate Reader; LabX, Switzerland), and the corresponding optical density ratio was expressed as cell vitality.

Cell apoptosis

The 2×10⁵ cells were collected in 1.5 mL EP tubes. The supernatant was discarded after centrifugation at 2000 × g at 4°C. Then, 500 μL of binding buffer was added to suspend the cells, and 5 μL of Annexin V-FITC was added and incubated at 4°C for 30 min in the dark. Then, 5 μL of propidium iodide (PI) was added, gently mixed and incubated at room temperature for 5 min. The cell apoptosis rates were detected using an Annexin-V-FITC detection kit (K201-100, BioVision, USA) and flow cytometry (version 10.0, FlowJo, FACS CaliburTM, BD, USA).

Luciferase assay

The sequences containing the wild-type (WT) or mutated (Mut) region MBTD1 were synthesized by Sangon (Shanghai, China) and inserted into a pGL3 vector (Promega Corporation, Madison, USA). For the luciferase reporter assay, miR-134-5p mimics and the respective reporter plasmids were transfected into cells using Lipofectamine 2000 according to the manufacturer’s protocol. After 24 h, the Renilla and Firefly luciferase activities were determined using the Dual-Luciferase Reporter Assay System (Promega Corp.) and a luminometer (Infinite 200 PRO NanoQuant; Tecan Group Ltd., Männedorf, Switzerland).

Tumour burden assay

Specific pathogen-free (SPF) 4-week-old BALB/c nude mice were purchased from the animal centre of Air Force Medical University (Shanghai, China). Saos-2 and U2OS cells were divided into three groups: vector-NC + sh-NC, vector-TTN-AS1 + sh-NC and vector-TTN-AS1 + sh-MBTD1. The mice in each group were injected with 1×10⁶ cells correspondingly. Twenty-eight days after injection, the mice were sacrificed, and tumours were removed to be weighed and photographed. The animal experiment protocol was approved by the Animal Experimentation Ethics Committee of Tongren Hospital.

Statistical analysis

All analyses were conducted using Prism GraphPad 7.0 software. All variables were shown as the mean ± standard deviation (SD). Comparisons among multiple groups were performed using one-way analysis of variance (ANOVA). P < 0.05 was considered statistically significant.