GABPB1-AS1 Promotes the Development of Osteosarcoma by Targeting SP1 and Activating the Wnt/β-Catenin Pathway

In this study, the role of GABPB1-AS1 in osteosarcoma (OS) was analyzed. The expression of GABPB1-AS1 in different OS cell lines U2OS, HOS, MG63, and hFOB1.19 was detected. SiRNA GABPB1-AS1 was transfected with U2OS and HOS cell lines. The effects of GABPB1-AS1 silencing on proliferation, clonal formation, and migration of U2OS and HOS were detected by CCK-8 method, plate cloning method, and Transwell chamber. Western blot analysis was used to detect the protein levels of SP1, Wnt, β-catenin, c-Myc, and SOX2 in osteosarcoma cells. The binding relationship between GABPB1-AS1 and miR-199a-3p in OS cells was detected by a dual-luciferase reporter gene assay. Results showed that GABPB1-AS1 was higher in OS cells than that in hFOB1.19. Silencing GABPB1-AS1 inhibited the proliferation, clonal formation, migration, and epithelial-mesenchymal transformation of U2OS and HOS. There was a binding relationship between GABPB1-AS1 and miR-199a-3p in OS cells. GABPB1-AS1 mediated osteosarcoma cells via the SP1/Wnt/β-catenin signaling pathway. This study suggested that GABPB1-AS1 plays a carcinogenic role in OS through the SP1/Wnt/β-catenin signaling pathway through competitive binding and inhibition of miR-199a-3p.


Introduction
At present, a surgical resection combined with chemotherapy is often used to control tumor metastasis. erefore, it is very important to seek new therapeutic strategies and therapeutic targets [1].
lncRNA can act as an oncogene or tumor suppressor to regulate the growth and proliferation of cancer cells [2]. lncRNAs and microRNAs (miRNAs) can regulate each other and affect the EMT of tumors [3]. Some lncRNAs play the role of tumor biomarkers in the diagnosis, treatment, and prognosis of osteosarcoma [4].
MicroRNAs are a class of noncoding RNAs (ncRNAs) about 22 nt in length. miRNAs are widely distributed, and their functions almost involve all tumor-related processes, including proliferation, differentiation, apoptosis, metastasis, angiogenesis, and immune response. e mechanism of action of microRNA is relatively simple. It binds to the target gene mRNA3'-untranslated region through complete or partial complementation and regulates the expression of a target gene by degrading or inhibiting mRNA translation. MicroRNA-199a-3p (miR-199a-3p) is one of many microRNAs. Studies [5,6] have observed its role in liver cancer, ovarian cancer, and osteosarcoma. However, it is rarely studied in osteosarcoma.
SP1 is widely present in tissue nuclei [7]. e SP1 protein functions mainly through the TGF-β classical signaling pathway, ERK, PI3K/Akt, and Wnt/β-catenin signaling pathway [8,9]. SP1 can regulate the transcriptional activity and expression of angiogenesis-related factors, oncogenes, and so on [10]. SP1 promotes tumor growth, metastasis, and inhibits apoptosis of tumor cells [11]. Further studies on the role of SP1 and its related signaling pathways in osteosarcoma will be helpful to further understand the mechanism of osteosarcoma occurrence and metastasis [12,13]. It also provides better and new molecular intervention targets and approaches for the targeted gene therapy of osteosarcoma.
In this study, the expression and effects of GABPB1-AS1 on the clonal formation, migration, and epithelial-mesenchymal transformation of OS cell lines were analyzed. Meanwhile, the binding sites of GABPB1-AS1 and miR-199a-3p and the effects of overexpression of GABPB1-AS1 and upregulation of miR-199a-3p on the Wnt/β-catenin signaling pathway were investigated.
is study provides reference for the treatment of OS.

CCK-8.
e cell concentration of each group was adjusted to 3 × 10 4 cells/mL. A 100 µL cell suspension was inoculated on 96-well plates. 10 µL of CCK-8 solution was added after incubating in an incubator for 72 h. e culture was continued for 2 hours, and the optical density (OD) value at 450 nm was measured on the microplate.

FISH.
e cell slipper was placed at the bottom of the 24-well plate, 5 × 10 3 /well. After 24 h culture, the supernatant was removed and the cells were cleaned with 1 × PBS. After fixation with 4% paraformaldehyde, PBS containing 0.5% Triton X-100 was added. e prehybridization solution was closed at 37°C, the lncRNA GABPB1-AS1 probe was hybridized overnight at 37°C, and the cells were cleaned with a hybridization lotion at 42°C under dark conditions. In the hybridization area of DAPI staining sections, the slides were fixed on the slides with sealing tablets under dark conditions. Experimental specimens were observed under laser confocal microscopy.

Clone Formation Experiment.
Cells of each group were collected and cultured for 24 h. After trypsin digestion, a suspension with a concentration of 1 × 10 6 cells/L was prepared. e 6-well plates were inoculated with 1 mL in each well and incubated at 37°C with 5% CO 2 for 14 d. e culture was terminated when visible clones were formed (the number of cloned cells was about 100). After discarding the medium and washing with PBS two times, 1 mL methanol was added to each well and fixed for 15 min. en the cells were rinsed slowly with running water, and 1 mL Giemsa dye was added to each well for 30 min. e cells were washed slowly with water and dried in a fume hood. e cells were counted under a microscope and the relative levels of cell cloning were calculated.

Transwell
Assay. Cells were taken from each group, and the cell suspension was prepared using a DMEM/F12 medium without FBS. 100 µL cell suspension (1 × 10 8 cells/mL) was inoculated into the upper compartment. e upper chamber was prepared with Matrigel in advance and cultured at 37°C for 24 h. e lower chamber was fixed. 0.1% crystal violet was dyed for 40 min at room temperature. e excess dye was washed and dried, and the cells passing through the microporous membrane were observed and counted under an optical microscope, and the relative level of cell migration was calculated.

Scratch Test.
e cells were cultured to the logarithmic stage and digested with 0.25% trypsin. With the help of a dropper, single cell suspension was blown, cell density was adjusted to 1 × 108/L, and the cell suspension was inoculated on a 6-well plate. Each well was placed at 1 mL under the conditions of 37°C, 5% CO 2 , and saturated humidity. e cells were cultured in the cell incubator for 24 h until the cells were basically fused 6-well plate was scratched vertically using a 100 μL micropipette head. e cells were rinsed twice with PBS solution.
e culture plates were placed in an incubator at 37°C with 5% CO 2 for 12, 24, and 48 h. ey were observed by an inverted phase-contrast microscope. In each field, 3 regions were randomly selected and the length of migration scratch space in 100× field was calculated.

qRT-PCR.
A RNA extraction kit was used to extract total RNA from various cells. e reverse transcription kit was used to obtain the cDNA and it was stored in a refrigerator at −20°C until use. Reaction procedure: 94°C 10 min; 94°C 30 s, 62°C 30 s, 62°C 30 s, 40 cycles. e reaction system refers to the qPCR kit instructions. β-Actin was used as an internal reference. e 2 −ΔΔCt method was used to calculate the relative expression levels of target genes in different cells.

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Journal of Oncology 2.9. Western Blot. Total protein was extracted from each group by a protein extraction kit. A BCA protein quantification kit was used for protein quantification. Protein were separated on 8% SDS-PAGE gels, transferred onto PVDF membranes (Bio-Rad, Hercules, CA, USA), and blocked for 1 h at room temperature. Monoclonal antibodies of SP1, Wnt, β-catenin, c-Myc, SOX2, and GAPDH mice diluted at 1 : 1000 were incubated overnight at 4°C. e film was washed with a TBST buffer and incubated with HRP-labeled goat anti-mouse IgG secondary antibody (1 : 2000) at room temperature for 2 h. e membrane was washed and color was developed with an ECL kit. e comprehensive gel imaging analysis system was utilized to capture pictures. Using GAPDH as an internal reference, the protein expression levels of each group were analyzed.

Statistical Analysis.
Statistical analyses were used SPSS 21.0 software. Each group was set up with 3 parallel experiments, which were repeated 3 times. e data were expressed as mean ± standard deviation. e comparison between the two groups was performed by t-test. One-way ANOVA was used for comparison between multiple groups, and the SNK-Q test was used for further pairwise comparison. p < 0.05 was considered statistically significant.

Distribution and the Expression Level of GABPB1-AS1.
e relative expression levels of GABPB1-AS1 in hFOB1.19, U2OS, HOS, and MG63 cell lines were analyzed by qPCR. GABPB1-AS1 in each OS cell line was significantly higher than that in normal osteoblast hFOB1.19 cell line (Figure 1(a)). Among them, the relative expression level of GABPB1-AS1 was higher in U2OS and HOS, so U2OS and HOS cell lines were selected for follow-up studies. Fluorescence in situ hybridization and nucleocytoplasmic separation confirmed that GABPB1-AS1 was mainly distributed in the cytoplasm (Figures 1(b)and1(c)).
3.6. Osteosarcoma Cells Were Mediated by GABPB1-AS1 via the SP1/Wnt/β-Catenin Signaling Pathway. Western blot was used to detect the protein levels of SP1, Wnt, β-catenin, c-Myc, and SOX2 in osteosarcoma cells with or without GABPB1-AS1 silencing. e results showed that silencing GABPB1-AS1 decreased the protein levels of SP1, Wnt, β-catenin, c-Myc, and SOX2 (Figure 6(a)). e experimental results of Figure 6(b) showed that overexpression of GABPB1-AS1 could upregulate SP1 expression. Clonal formation to evaluate the salvage effect of LiCl treatment on proliferation of GABPB1-AS1-silenced osteosarcoma cells [14]. Compared with the sh-GABPB1-AS1 group, the clone formation ability of the sh-GABPB1-AS1 + LiCl group was enhanced (Figure 6(c)). e wound healing assay and Transwell assay were used to evaluate the effects of LiCl treatment on the migration and invasion of GABPB1-AS1silenced osteosarcoma cells. Compared with the sh-GABPB1-AS1 group, sh-GABPB1-AS1 + LiCl group showed enhanced cell migration and invasion ability (Figures 6(d) and6(e)). In U2OS and HOS cells, the expression of E-cadherin was decreased in the sh-GABPB1-AS1 + LiCl  Journal of Oncology group compared with the sh-GABPB1-AS1 group. In contrast, N-cadherin, Slug, and Twist1 expression levels increased (Figures 6(f )-6(i)). ese results indicate that GABPB1-AS1 mediated the EMT process of osteosarcoma cells through SP1/Wnt/β-catenin signaling pathway.

Discussion
e proliferation and local invasion of osteosarcoma cells are the key stages of tumorigenesis and development [15]. Exploring the molecular mechanism of proliferation and migration of osteosarcoma for identifying new therapeutic targets and developing new therapeutic strategies was important [16,17].
ere are a large number of lncRNAs with an abnormal expression in osteosarcoma tissues, which play an important role in transcription and posttranscription levels [18]. lncRNAs regulate the proliferation, apoptosis, invasion, migration, drug resistance, and other malignant biological behaviors of osteosarcoma [19,20]. Zhu et al. [21] analyzed 5 pairs of osteosarcoma and paracancer tissues by lncRNA microarray and screened differentially expressed lncRNAs. Results showed that 65 lncRNAs were upregulated and 13 lncRNAs were downregulated. It was further found that lncRNA nucleolar small RNA host gene 16 (ENSG00000163597) had the highest expression level in osteosarcoma. Studies showed that host gene 16 of small nucleolar RNA (ENSG00000163597) can act as an endogenous sponge for miR-205, upregulating the expression of zinc finger e-box zinc finger protein 1 and significantly enhancing the proliferation ability of osteosarcoma cells. Li et al. [22] found that lncRNA actin fibro-associated protein 1 (AFAP1)-AS1 expression was significantly upregulated in osteosarcoma tissues and cell lines. AFAP1-AS1 can capture miR-4695-5p through sponging, thus upregulating the expression of transcription factor 4. Transcription factor 4, as a key transcription factor in the Wnt/β-catenin pathway, promotes the proliferation of osteosarcoma.
GABPB1-AS1 in osteosarcoma tissues was significantly higher than that in adjacent tissues. e high expression of GABPB1-AS1 may be related to the progression of osteosarcoma. A functional loss assay showed that the survival rate, clonal formation, and migration of U2OS and HOS cells were decreased after the GABPB1-AS1 expression was inhibited by transfection with GABPB1-AS1 small interfering RNA. E-cadherin can maintain epithelial cell morphology and tissue integrity. Conversely, it promotes metastasis and diffusion of tumor cells. Studies have shown that lncRNA TTTYl5 knockdown can reduce the invasion ability of osteosarcoma cells. N-cadherin expression was decreased and E-cadherin was significantly increased in U2OS and HOS cells after GABPB1-AS1 expression was inhibited, which was consistent with the results of functional loss experiment. ese results suggest that GABPB1-AS1 plays an oncogene role in osteosarcoma. In recent years, a number of studies have shown that lncRNAs can play a role in tumors by targeting miRNA and blocking its function. For example, FGD5-AS1 regulates the growth, migration, invasion, and apoptosis of esophageal squamous cell carcinoma by targeting miR-383 [23]. FGD5-AS1 increases the ability of malignant proliferation, migration, and invasion of colorectal cancer cells by interacting with miR-302e [24].
is study found that miR-199a-3p may interact with GABPB1-AS1. miR-199a-3p is a member of the miR-family, and previous studies have shown that miR-199a-3p has a low expression in OS [25]. miR-199a can inhibit the of nonsmall-cell lung cancer by regulating HIF-1α [26]. miR-199a-3p can induce metastasis of malignant tumor cells by inhibiting the expression of SMARCA2, downregulating the expression of SMARCA4, and inhibiting the expression of TGF-β. TGF-β also regulates epithelial-mesenchymal transformation and participates in tumor cell metastasis [27].
In this study, dual-luciferase assay confirmed that GABPB1-AS1 had a negative regulatory effect on miR-199a-hsa-miR-199a-3p GABPB1-AS1 Target: miRNA : (a) 3p. In addition, the inhibition of GABPB1-AS1 expression could increase miR-199a-3p. Further functional analysis showed that overexpression of miR-199a-3p enhanced inhibition of GABPB1-AS1 on proliferation, migration, apoptosis, and related protein expression of U2OS and HOS cells. Inhibition of miR-199a-3p expression reversed the effects of GABPB1-AS1 inhibition on proliferation, migration, apoptosis, and related protein expression of U2OS and HOS cells.
e abovementioned studies indicate that the GABPB1-AS1/miR-199a-3p pathway in osteosarcoma and the exploration of downstream target genes and possible signaling pathways of miR-199a-3p is the next research focus. e occurrence and metastasis of tumors are highly correlated with abnormal activation of the Wnt signaling pathway [28]. In particular, lncRNA dysregulation can activate Wnt signal transduction pathways, leading to cancer growth and metastasis. Eight members of the SP family  Journal of Oncology 9 (SP1∼SP8) are a highly evolutionarily related transcription factor [29]. SP1 was first discovered in this family, and its gene, located at 12q1, is widely expressed in various tissues. SP1 affects the malignant tumors from different tissues [30].
In this study, we found that GABPB1-AS1 promotes the development of osteosarcoma by targeting SP1 to activate the Wnt/β-catenin pathway.

Conclusion
In summary, GABPB1-AS1 was highly expressed in osteosarcoma. e inhibition of GABPB1-AS1 can reduce the proliferation and migration of osteosarcoma cells. Mechanism studies showed that lncRNA GABPB1-AS1, as a competitive endogenous RNA, upregulated the expression of SP1 and promoted the Wnt/β-catenin pathway through sponge adsorption of miR-199a-3p, ultimately promoting the proliferation and malignant evolution of osteosarcoma.
Data Availability e analyzed datasets generated during the study are available from the corresponding author on reasonable request.

Conflicts of Interest
e authors declare that they have no conflicts of interest.