Silencing of the Long Non-Coding RNA TTN-AS1 Attenuates the Malignant Progression of Osteosarcoma Cells by Regulating the miR-16-1-3p/TFAP4 Axis

Objectives Osteosarcoma (OS) is a type of bone malignancy. This study attempted to explore the effect of long non-coding RNA TTN-AS1 (TTN-AS1) on OS and to determine its molecular mechanisms. Methods The expression of TTN-AS1, microRNA-16-1-3p (miR-16-1-3p), and transcription factor activating enhancer binding protein 4 (TFAP4) in OS was assessed using qRT-PCR. The OS cell proliferation, migration, and invasion were measured using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), wound-healing, and transwell assays. N-cadherin and MMP-2 protein level was determined with western blot. Interactions between TTN-AS1 and miR-16-1-3p or TFAP4 and miR-16-1-3p were confirmed using the dual-luciferase reporter assay. Additionally, an OS xenograft tumor model was constructed to assess the effect of TTN-AS1 on tumor growth. Results TTN-AS1 and TFAP4 expression was increased in OS, while miR-16-1-3p expression was decreased. TTN-AS1 silencing restrained OS cell proliferation, migration, invasion, N-cadherin and MMP-2 protein expression, and hindered tumor growth. MiR-16-1-3p overexpression retarded the malignant behavior of OS cells. TTN-AS1 played a carcinostatic role by down-regulating miR-16-1-3p in the OS cells. Moreover, miR-16-1-3p inhibition or TFAP4 elevation weakened the suppressive effect of TTN-AS1 silencing on OS cell tumor progression. Conclusion TTN-AS1 promoted the proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) of OS cells via mediating the miR-16-1-3p/TFAP4 axis. TTN-AS1 may be a critical target for improving OS.


INTRODUCTION
Osteosarcoma (OS) is a malignancy of the bone that generally occurs in adolescents and children (1). It is characterized by a high level of complexity and heterogeneity (2). Resection surgery, combined with polychemotherapy, is an important therapeutic approach for its treatment (3). The metastasis rate of OS is over 30%, with high recurrence and poor efficacy (4). OS progression is complex and is caused by aberrant oncogenes or anti-oncogenes expression (5,6). Therefore, it is imperative to investigate the pivotal molecules involved in the malignant progression of OS.
Herein, we demonstrated the role of TTN-AS1 in the tumor progression of OS. This study confirmed the relationships among TTN-AS1, miR-16-1-3p, and TFAP4, thus providing a new direction for OS treatment.

Tissue Samples
Sixty-three patients with OS were recruited from our hospital between April 2016 and January 2018. OS tissues and adjacent tissues were harvested. Patients had never received anti-cancer treatment. This study was permitted by ethics committee of our hospital in accordance with the Declaration of Helsinki, and informed consent was obtained from each patient and guardian.

Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)
Total RNA was extracted from tissues and cells using the TRIzol reagent (Invitrogen). cDNA samples were obtained by reverse transcription using the PrimeScript RT Reagent kit (Takara, Tokyo, Japan). The QuantiTect SYBR Green PCR kit (Takara) was used for qRT-PCR analysis. qRT-PCR was performed using a 7500 Real-time PCR system (Applied Biosystems, Foster City, CA, USA) with the following reaction conditions: initial denaturation at 95°C for 10 min, followed by 40 cycles at 95°C for 10 s, 60°C for 20 s, and 72°C for 34 s. The relative expression was calculated using the 2 -DDCq method. GAPDH, U6, and bactin were used to normalize TTN-AS1, miR-16-1-3p, and TFAP4, respectively. Primer sequences are listed in Table 1.

Wound Healing Assay
The OS cells (5 × 10 5 cells/well) were incubated in 6-well plates until reached about 100% confluence. Then, the cell monolayer was wounded with a pipette tip and the plates were washed twice with phosphate-buffered saline (PBS) to remove detached cells. The cells were then cultured in serum-free DMEM (Invitrogen) at 37°C. During the following 24 h, the cells migrated into the wound area. Cell migration images were captured using an inverted microscope (Olympus, Tokyo, Japan).

Transwell Assay
The OS cells (2 × 10 5 ) in serum-free DMEM (Invitrogen) were added to the upper chambers pre-coated with matrigel (Sigma). DMEM, containing 10% FBS, was added to the lower chambers. Cells remaining in upper chamber were wiped with cotton swab after incubation for 48 h. Cells in lower chambers were fixed with 4% paraformaldehyde for 30 min and stained with 0.1% crystal violet for 30 min. Finally, the images were counted using an inverted microscope (Olympus).

Tumor Formation
Total 12 male BALB/c nude mice were gained from HFK Bioscience Co., Ltd (Beijing, China). The mice were housed at 22-26°C in an atmosphere of 55-65% relative humidity and fed a normal diet. MG63 cells (1 × 10 6 cells) expressing sh-NC or sh-TTN-AS1-1 were subcutaneously injected into the flank region of nude mice to establish OS xenograft model (n = 6 per group). Tumor volumes (0.5 × length × width 2 ) were assessed on a weekly basis. Four weeks after injection, mice were anesthetized (pentobarbital sodium, 50 mg/kg), sacrificed through cervical dislocation. Finally, intact tumors were exfoliated and weighted. All the animal experiments were approved by the Animal Care and Use Committee of our hospital.

RNA Immunoprecipitation (RIP) Assay
RIP assay was performed using an EZ-Magna RIP kit (Millipore), following the manufacturer's instructions to further confirm the target relationship between TTN-AS1 and miR-16-1-3p. OS cells at 85% confluence were lysed in RIP lysis buffer, then the cell extract was incubated with magnetic beads and an antibody against argonaute 2 (Anti-AGO2; ab32381; Abcam, Cambridge, MA, USA) or immunoglobin G (Anti-IgG; ab109761; Abcam) at 4°C overnight. Proteinase K was used to digest the protein and immunoprecipitated RNAs were isolated. Finally, purified RNAs were subject to qRT-PCR analysis.

Bioinformatics-Based Prediction and Analyses
We used predicting tool, LncBase, for prediction of the miRNAs that target TTN-AS1. MiR-16-1-3p was predicted as one of the candidate miRNAs. MiR-16-1-3p possesses strong tumor suppressive and anti-metastatic properties in OS (23). However, the detail regulatory mechanism between TTN-AS1 and miR-16-1-3p in OS remains unknown. We therefore selected miR-16-1-3p for subsequent studies. We additionally employed the online tool, targetScan, for predicting the possible target genes of miR-16-1-3p. TFAP4 was predicted as one of the candidate mRNAs. TFAP4 is a well-known oncogene, which up-regulated in various cancers (27,28,30). However, the detail regulatory mechanism between miR-16-1-3p and TFAP4 in OS remains unknown. We therefore selected TFAP4 for subsequent studies.

Statistical Analysis
All statistical analyses were performed using GraphPad Prism 7.0 (GraphPad Software, La Jolla, CA, USA). Data are presented as mean ± standard deviation (SD). The differences between two groups or among multiple groups were analyzed using the Student's t-test or one-way ANOVA followed by Tukey's post-hoc test. The significance of the correlations was determined using Pearson's correlation analysis. P values < 0.05 were considered as statistically significant.

TTN-AS1 Expression Was Enhanced in Human OS Tissues
qRT-PCR was performed to confirm whether TTN-AS1 was differentially expressed in OS tumor. Results displayed that TTN-AS1 expression was dramatically enhanced in tumor tissues of OS patients (P < 0.001, Figure 1A). Additionally, TTN-AS1 expression was higher in tissues from patients in TNM III/IV (P < 0.001, Figure 1B). TTN-AS1 expression was significantly up-regulated in the metastatic tumors (P < 0.001, Figure 1C). As displayed in Table 2, TTN-AS1 expression was positively correlated with the degree of metastasis (P < 0.05), and WHO grade (P < 0.01) in OS patients.

TTN-AS1 Knockdown Repressed the Progression of OS
We used RNA interference approaches to investigate whether knockdown of TTN-AS1 affects the progression of OS. TTN-AS1 expression was higher in the HOS, MG63, and U2OS cells than that in the hFOB cells (P < 0.01, Figure 2A). MG63 and U2OS cells (OS cells) were selected for subsequent experiments because of their high TTN-AS1 expression. TTN-AS1 expression was markedly decreased by the transfection of sh-TTN-AS1-1, -2, and -3 into OS cells (P < 0.01, Figure 2B). Sh-TTN-AS1-1 was used for subsequent experiments due to its relatively high silence efficiency. Subsequently, sh-TTN-AS1-1 considerably decreased OS cell proliferation after 72 (P < 0.05) and 96 h (P < 0.01, Figure 2C) of culture. Moreover, TTN-AS1 down-regulation notably reduced invasion and migration of OS cells (P < 0.01, Figures 2D, E). TTN-AS1 silencing markedly reduced the tumor volume and weight in mice (P < 0.05, Figures 2F, G).

DISCUSSION
Up-regulation of lncRNAs, such as lncRNA BCAR4 (31), lncRNA TUG1 (32), and lncRNA MF12 (33) has been shown to exert a pivotal influence on OS pathogenesis. Here, TTN-AS1 expression was increased in tumor tissues of OS patients, and was associated with WHO grade and metastasis in OS patients. The function of TTN-AS1 was found to be similar to that of some lncRNAs in OS. The enhanced expression of lncRNA TP73-AS1 is related to distant metastasis and predicts poor outcome in OS patients (34). LncRNA MALAT1 is overexpressed in OS and is clearly correlated with distant metastasis and advanced clinical stage (35). Above all, we suggested that TTN-AS1 expression may be related to OS progression. TTN-AS1 acts as an oncogene in various malignancies. TTN-AS1 sponges miR-142-5p to modulate CDK5, triggering the growth and metastasis of lung adenocarcinoma (36). TTN-AS1 mediates miR-153-3p to accelerate malignant behaviors by mediating ZNRF2 in papillary thyroid cancer (37). TTN-AS1 suppression hinders the progression of ovarian cancer by upregulating miR-139-5p (38). Notably, TTN-AS1 binds with miR-134-5p to increase MBTD1 expression, elevate OS cell viability, and inhibit cell apoptosis (17). In this study, TTN-AS1 silencing restrained the OS cell tumor progression. Additionally, some lncRNAs knockdown suppressed tumor growth in OS. For examples, inhibition of lncRNA TUG1 elevates miR-9-5p expression and attenuates the growth of OS tumor xenografts (39). Knockdown of lncRNA miR210HG reduces the OS tumor volume and weight in mice (40). LncRNA TAB silencing  This phenomenon may be attributed to that the size of primary OS in human can be influenced by massive factors in vivo, and the detailed mechanisms leading to this situation still needs to be studied. An increasing number of studies have shown that lncRNAs influence tumor progression by working as sponges or competing endogenous RNAs (ceRNAs) of miRNAs. The above-mentioned results demonstrated that TTN-AS1 knockdown attenuates OS cell tumor progression by increasing miR-16-1-3p expression. TFAP4 is a direct transcriptional target of certain miRNAs and participates in the tumor progression of diverse tumors. For examples, miR-302c attenuates cell EMT and metastasis through decreasing TFAP4 expression in colorectal cancer (49). MiR-608 induces cell apoptosis by targeting TFAP4 in NSCLC (50). LncRNA LINC00520 interacts with miR-520f-3p to promote the malignant behaviors of glioma cells through targeting TFAP4 (29). TFAP4 is up-regulated and serves as an oncogene in various cancers, such as gastric cancer (27), neuroblastoma (25), and hepatocellular carcinoma (51). Here, TFAP4 expression was elevated in OS cells and was inversely correlated with miR-16-1-3p expression. Given the relationship between TTN-AS1 and miR-16-1-3p, we hypothesized that TTN-AS1 knockdown may inhibit TFAP4 by up-regulating miR-16-1-3p in OS cells. Encouragingly, the feedback experiments showed that up-regulation of TFAP4 Taken together, we suggest that sh-TTN-AS1-1 exerts its antitumor role through mediating miR-16-1-3p/TFAP4 axis in OS.

Interactions between miRNAs and lncRNAs, including
Moreover, previous studies have demonstrated that some other TTN-AS1 related axes were also involved in the regulation of OS. For instances, TTN-AS1 knockdown inhibits OS cell proliferation, migration, and invasion and induces apoptosis via targeting the miR-376a/dickkopf-1 axis (16). Down-regulation of TTN-AS1 not only decreases OS cell viability and drug resistance, but also prompts cell apoptosis by regulating the miR-134-5p/MBTD1 axis (17). Above all, we speculated that there are many other downstream targets of TTN-AS1 that have not yet been determined in OS. Related exploration will be considered in our future studies. Collectively, our results indicated that TTN-AS1 expression was elevated in OS. TTN-AS1 increased TFAP4 expression through competition with miR-16-1-3p, thus playing an oncogenic role in OS. Additionally, TTN-AS1 deficiency attenuated the OS cell proliferation, migration, invasion, and EMT by regulating the miR-16-1-3p/TFAP4 axis. Findings of the current study may provide a promising therapeutic target for OS.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding authors.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by Ethics Committee of Jinan Central Hospital, Cheeloo College of Medicine, Shandong University. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.
to be published. ZZ and LC: data acquisition and drafting the article. XW and QZ: data acquisition and final approval of the version to be published. SL: Data analysis and interpretation, and drafting the article. All the authors took part in the experiment.