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

Aim: To explore the mechanism by which long non-coding RNA (lncRNA) TTN-AS1 regulates osteosarcoma cell apoptosis and drug resistance via the microRNA miR-134-5p/malignant brain tumour domain containing 1 (MBTD1) axis. Results: The lncRNA TTN-AS1 was highly expressed in osteosarcoma and was associated with poor prognosis. The lncRNA TTN-AS1 promoted cell viability and inhibited apoptosis. MiR-134-5p targeted MBTD1, which was regulated by lncRNA TTN-AS1. MBTD1 was highly expressed in osteosarcoma and was associated with poor prognosis. MBTD1 promoted cell viability and inhibited apoptosis, and knockdown of MBTD1 reversed the cancer-promoting effects of lncRNA TTN-AS1. Downregulation of lncRNA TTN-AS1 reduced drug resistance. Conclusion: In osteosarcoma, lncRNA TTN-AS1 promoted the expression of MBTD1 by targeting miR-134-5p and regulated cell growth, apoptosis and drug resistance. Methods: The expression characteristics of genes in osteosarcoma patients were analysed using bioinformatics. Plasmid transfection technology was applied to silence or overexpress lncRNA TTN-AS1, miR-134-5p and MBTD1. Western blotting and quantitative polymerase chain reaction (qPCR) were used to detect protein and RNA, respectively. A cell counting kit 8 (CCK-8) and flow cytometry were used to detect cell viability and apoptosis. The effects of lncRNA TTN-AS1 and MBTD1 on osteosarcoma in vivo were studied by using a tumour burden assay.


INTRODUCTION
Osteosarcoma is a musculoskeletal malignancy that originates in the interstitial cell line [1]. The incidence of osteosarcoma is approximately 3 per million, and the incidence of osteosarcoma in men is higher than that in women [2]. The predilection site of osteosarcoma is the medullary ends of the long bones, especially the distal femur and the proximal humerus around the knee joint [3]. The course of osteosarcoma progresses rapidly, and morbidity and mortality are high [4,5]. Currently, the main treatments for osteosarcoma include surgery, chemotherapy, immunotherapy and gene therapy [6][7][8]. The strong proliferative capacity and drug resistance of osteosarcoma cells are important factors affecting prognosis [9,10].
Long non-coding RNA (lncRNA) typically has a length of more than 200 nucleotides [11]. LncRNAs do not have protein translation capabilities, but they play an important role in various processes by directly or indirectly regulating protein expression [12]. LncRNAs can not only directly participate in the regulation of gene expression but can also act as competing endogenous RNAs (ceRNAs) to interact with miRNAs [13,14]. A microRNA (miRNA) is an evolutionarily conserved single-stranded RNA that typically contains 21-24 nucleotides [15]. MiRNAs play a key role in the post-transcriptional regulation of mRNA by targeting the 3′ untranslated region (UTR) of mRNA, leading to mRNA degradation or translational inhibition [15]. In recent years, many studies have found that lncRNA is involved in the development and progression of osteosarcoma as ceRNA [16][17][18]. The number of single lncRNAs is large, and the pathways are diverse. Bioinformatics helps to screen for meaningful lncRNAs [19,20].
In this paper, we found that the lncRNA TTN-AS1 and malignant brain tumour domain containing protein 1 (MBTD1) were overexpressed in osteosarcoma, and they were associated with the poor prognosis of osteosarcoma patients. Based on this, we revealed a novel mechanism by which lncRNA TTN-AS1 regulated proliferation and apoptosis of osteosarcoma cells via miR-134-5p/MBTD1. This may provide new ideas for the discovery of strategies for osteosarcoma treatment.

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).

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  ( 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.

DISCUSSION
In this study, we discovered the role of lncRNA TTN-AS1 and MBTD1 in osteosarcoma and analysed the The role of lncRNA in osteosarcoma has been confirmed, and both lncRNA and miRNA can be used as markers for the diagnosis and prognosis of osteosarcoma [21][22][23]. This study found that lncRNA TTN-AS1 was highly expressed in osteosarcoma patients and was associated with poor prognosis. TTN-AS1 is located on chromosome 2: 178,521,183e178,779,963 and was first discovered to be associated with oesophageal squamous cell carcinoma. Patients with high expression of lncRNA TTN-AS1 have a worse prognosis and a higher risk of metastasis [24]. The results of Luo's study [25] also show that lncRNA TTN-AS1 is overexpressed in lung adenocarcinoma and promotes tumour development by modulating the PTEN/PI3K/AKT pathway. Recent studies show that in cervical cancer, gastric cancer and thyroid cancer, lncRNA TTN-AS1 can act as a ceRNA to promote cancer by targeting miRNA [26][27][28]. However, the mechanism of action of lncRNA TTN-AS1 in tumours is unclear, and there is no research on lncRNA TTN-AS1 in osteosarcoma. The results of this study showed that lncRNA TTN-AS1 could promote cell viability and inhibit apoptosis, and downregulation of lncRNA TTN-AS1 could reduce drug resistance. This revealed the pro-cancer effects of lncRNA TTN-AS1 on osteosarcoma and the potential for involvement in osteosarcoma drug resistance.
To further explore the mechanism by which lncRNA TTN-AS1 regulated osteosarcoma cell growth and apoptosis, we found through bioinformatics prediction and experimental studies that lncRNA TTN-AS1 targeted miR-134-5p levels as ceRNA and that miR-134-5p inhibited MBTD1 expression by directly binding to the 3′UTR region of MBTD1 mRNA. Studies have found anti-tumour mechanisms of miR-134-5p in non-small cell lung cancer and nasopharyngeal carcinoma [29][30][31].
MiR-134-5p can also be targeted and regulated by lncRNA [32,33]. Studies have found that miR-134-5p exerts anti-tumour effects by affecting target gene expression in osteosarcoma [34][35][36]. This study demonstrated that miR-134-5p was regulated by lncRNA TTN-AS1 to inhibit osteosarcoma growth and apoptosis.   MBTD1 belongs to the polycomb group (PcG) and regulates the transcription process. This study found the role of MBTD1 in tumourigenesis [37]. MBTD1 is associated with low-grade endometrial cancer [38]. However, the role of MBTD1 in osteosarcoma has not been studied. In this study, MBTD1 was highly expressed in osteosarcoma, and high levels of MBTD1 were closely related to malignancy and poor prognosis of osteosarcoma. Cell experiments confirmed that MBTD1 could promote cell viability and inhibit apoptosis. Both the cell experiments and animal experiments confirmed that downregulating the level of MBTD1 reversed the effects of overexpression of lncRNA TTN-AS1 on tumour growth.
In conclusion, lncRNA TTN-AS1 promoted the expression of MBTD1 by targeting miR-134-5p and regulated cell growth, apoptosis and drug resistance in osteosarcoma. However, further research is needed on the mechanisms by which TTN-AS1 participates in drug resistance and by which MBTD1 promotes the development of osteosarcoma.

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.

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 × 10 4 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

Cell apoptosis
The 2×10 5 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 1×10 6 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.