THAP9-AS1 Promotes Tumorigenesis and Reduces ROS Generation through the JAK2/STAT3 Signaling Pathway by Increasing SOCS3 Promoter Methylation in Osteosarcoma

Increasing studies have demonstrated that dysfunction of long noncoding RNAs (lncRNAs) plays critical roles in the development of human cancers. THAP9-AS1 has been reported to be dysregulated and associated with tumor progression in some cancers. However, the function and mechanism of THAP9-AS1 in osteosarcoma (OS) remain unclear. In the present study, we found that the expression of THAP9-AS1 was significantly upregulated in OS tissues and associated with the advanced stage of tumors and poor prognosis of patients. Blast comparison results showed that the SOCS3 promoter region and THAP9-AS1 had base complementary pairing binding sites. The interactions between THAP9-AS1, DNA methyltransferases (DNMTs), and SOCS3 were assessed by RIP and ChIP assays. The results of methylation-specific PCR (MSP) and bisulfite sequencing PCR (BSP) validated that THAP9-AS1 enhanced the methylation level of the SOCS3 promoter. The mRNA levels of SOCS3 in OS cells could be reversed by the demethylation agent 5-aza-2′-deoxycytidine. The mRNA expression of SOCS3 was downregulated in OS tissues and negatively correlated with THAP9-AS1 expression in tumors. Moreover, the western blot and immunofluorescence (IF) assay data showed that THAP9-AS1 activated the JAK2/STAT3 signaling pathway by upregulating p-JAK2 and p-STAT3 and the nuclear translocation of p-STAT3. Functionally, ectopic expression of THAP9-AS1 promoted cell proliferation, migration, and invasion and inhibited apoptosis, and this phenomenon could be reversed by SOCS3. Introduction of the JAK/STAT inhibitor AG490 partially abolished the stimulative effect of THAP9-AS1 on cellular processes. In addition, THAP9-AS1 decreased oxidative stress by reducing reactive oxygen species (ROS) and enhancing the mitochondrial membrane potential of OS cells via the SOCS3/JAK2/STAT3 pathway. Stable overexpression of THAP9-AS1 contributed to tumor growth and metastasis in vivo. In total, our findings suggested that upregulation of THAP9-AS1 might recruit DNMTs to epigenetically inhibit SOCS3, thereby activating the JAK2/STAT3 signaling pathway and oncogenesis of OS. These results provide novel insights for the understanding of OS progression.


Introduction
Osteosarcoma (OS) is one of the most common malignant bone tumors and occurs more frequently in long bones in children and adolescents [1]. Lung metastasis is often observed in many OS patients upon preliminary diagnosis, and the 5-year survival rate of these patients remains less than 20% [2]. Therefore, a greater depth of insight into the molecular mechanisms involved in OS is essential and useful for identifying new biomarkers and novel treatment strategies.
Long noncoding RNAs (lncRNAs), a group of noncoding RNAs, are longer than 200 nucleotides in length and lack protein coding capacity [3]. Dysregulation of lncRNAs is related to a wide range of tumor cellular processes, such as proliferation, apoptosis, invasion, autophagy, and angiogenesis [4]. Several lncRNAs were identified as potential diagnostic and prognostic markers of OS patients and acted as oncogenes or tumor suppressors in the development of OS. For example, TTN-AS1 increased cell growth and drug resistance by regulating miR-134-5p/MBTD1 [5]. KCNQ1OT1 acts as a competing endogenous RNA (ceRNA) to regulate ALDOA by sponging miR-34c and promotes the Warburg effect in OS cells [6]. Our previous study revealed that PVT1 contributed to glucose metabolism, cell proliferation, and motility through the miR-497/HK2 pathway in OS cells [7]. Another mechanism by which lncRNAs regulate gene expression and tumor progression is by regulating promoter DNA methylation through recruiting DNA methyltransferases (DNMTs) [8,9]. THAP9-AS1, a long noncoding RNA that is located on 4q21.22, was previously discovered to be upregulated in breast cancer by using next-generation deep sequencing [10]. THAP9-AS1 expression was positively correlated with advanced tumor stages and poor prognosis; knockdown of THAP9-AS1 suppressed cell proliferation and invasion and enhanced apoptosis in esophageal squamous cell carcinoma (ESCC) [11]. THAP9-AS1 was identified as a promotor of pancreatic ductal adenocarcinoma (PDAC) progression by activating YAP signaling, which in turn also modulates THAP9-AS1 transcription [12]. However, the function and mechanism of THAP9-AS1 in the development of OS remain unknown.
Aberrant JAK/STAT signaling has been demonstrated to contribute to cancer progression and metastatic development in various cancers. Activation of the JAK2/STAT3 signaling pathway is involved in LCP1-induced cell proliferation, migration, and invasion in OS [13]. Overexpression of PARK2 inhibited tumor growth, invasion, and angiogenesis, probably through suppressing the JAK2/-STAT3/VEGF signaling pathway in OS [14]. Targeting the JAK2/STAT3 pathway is currently one of the most promising therapeutic strategies in the treatment of OS [15,16]. Suppressors of cytokine signaling (SOCS) proteins can attenuate JAK/STAT signaling by interfering with the activity of JAK kinases [17]. However, the cross-talk between lncRNAs and the JAK2/STAT3 signaling pathway in OS progression remains largely unknown.
This study is aimed at investigating the biological function of THAP9-AS1 in OS and the underlying mechanisms. Our results mainly revealed that THAP9-AS1 was upregulated in OS and negatively correlated with the prognosis of patients. Overexpression of THAP9-AS1 promoted cell proliferation and metastasis in vitro and in vivo and reduced oxidative stress, likely by regulating SOCS3 methylation and expression and the downstream JAK2/STAT3 signaling pathway.

Clinical Samples and Cell
Lines. Forty pairs of OS tissues and their matched adjacent nontumor tissues (at least 3 cm away from the tumor) were collected from patients who underwent surgery at the Affiliated Hospital of Qingdao University between March 2015 and July 2016. This study was approved by the Research Ethics Committee of the Affiliated Hospital of Qingdao University. All tissue specimens were recognised by pathologists. Informed consents were obtained from patients. None of the patients received chemotherapy before surgery. Tissue samples were collected during surgery and stored at -80°C until use. The clinicopathological features of the patients were recorded and are summarized in Table 1.
Four human OS cell lines (143B, MG63, SaOS-2, and U2OS) and a normal human osteoblast cell line (hFOB1. 19) were obtained from the China Academy of Sciences (Shanghai, China) and maintained in a humidified incubator at 37°C with a 5% CO 2 atmosphere.
2.10. Cell Proliferation Assay. MTT method was used to evaluate cell proliferation. In brief, cells were seeded in 96-well plates (1 × 10 4 cells/well) and incubated at 37°C. At the indicated time points (0, 24, 48, and 72 h), the medium was removed, and MTT (20 μL; 5 mg/mL) was added to each well. After incubation for another 4 hours, DMSO was added to each well, and the absorbance was measured at 490 nm by a microplate reader (Bio-Tek, Germany).

Cell Apoptosis Assay.
A total of 2 × 10 5 cells were cultured in 6-well plates. After being harvested and resuspended in 100 μL of Annexin-V-binding buffer, the cells were incubated with Annexin-V-FITC and propidium iodide (PI, 1 μg/mL). After incubation for 15 minutes in the dark, the stained cells were measured using a BD FACS-Calibur (BD Bioscience, San Diego, CA, USA), and the analysis was performed through flow cytometry (BD Bioscience).

Cell Migration and Invasion
Assays. Transwell chambers were used to measure cell migration and invasion. The upper surface of the filter (8.0 μm; Biosciences, Heidelberg, Germany) with and without Matrigel coating (BD Biosciences) was used in invasion and migration assays, respectively. A total of 3 × 10 4 posttransfection OS cells in 100 μL serum-free DMEM were added to the upper chambers, and the lower chambers were filled with 700 μL DMEM with 20% FBS. After incubation for 24 h, the membranes were fixed with 4% methanol and stained with 0.1% crystal violet. The stained cells were counted in five random fields.

Analysis of Reactive Oxygen Species (ROS) and
Mitochondrial Membrane Potential. For detection of ROS generation, OS cells were seeded in a 24-well plate (1 × 10 5 cells/well). After being washed with PBS, the medium was replaced with 500 μL DCF-DA and incubated at 37°C for 20 min. After washing, the cells were observed under a fluorescence microscope. The mitochondrial membrane potential of cells was evaluated using the JC-1 probe staining method (Beyotime, China). In brief, the treated cells were seeded onto 6-well plates and cultured for 24 h. After washing with PBS and adding complete medium, the cells were immersed in JC-1 working solution and incubated at 37°C for 20 min. After the cells were washed with cold JC-1 staining buffer three times, the MMP of the cells was detected by fluorescence microscopy (Leica, Germany). Finally, the green/red fluorescence intensity ratio can indirectly reflect the state of the mitochondrion.
2.14. Immunofluorescence (IF) Staining. The transfected cells were fixed with 4% paraformaldehyde, washed three times with PBS, and permeabilized with 0.2% Triton X-100/PBS solution. Then, the cells were incubated with an anti-p-STAT3 antibody (ab76315) overnight at 4°C, washed with PBS, and incubated with a fluorescence-conjugated secondary antibody. After that, the cells were incubated with 4′,6diamidine-2′-phenylindole dihydrochloride (DAPI) for 3 min at room temperature. Fluorescent images were obtained using a confocal microscope (Olympus).

Xenograft Tumor Growth and In Vivo Metastasis
Assays. Animal experiments were approved by the Institutional Committee for Animal Research of the Affiliated Hospital of Qingdao University. Ten BALB/c nude mice were used in this research (n = 5 for each group), and 5 × 10 6 cells transfected with THAP9-AS1 or vector control cells were subcutaneously injected into the flanks of mice. Tumor volume (width 2 × length/2) was measured every five days. Finally, mice were sacrificed, and samples were processed for further research. For the in vivo metastasis assay, MG63 cells (1 × 10 7 ) expressing THAP9-AS1 or vector control were injected into the tail vein of BALB/c nude mice (n = 5/group). All mice were sacrificed after 49 days. The lung tissues were obtained and prepared into paraffinembedded sections. HE staining was performed to check the metastatic nodules of the lungs under a light microscope. At least five random sections per lung tissue were analyzed.
2.17. Immunohistochemical (IHC) Staining. The tumor tissues were paraffin-embedded and cut into 5 μm thick slides for immunohistochemical analysis. After incubating in 0.15% Triton X-100 and blocking with 1% goat serum albumin in modified D-PBS Tween-20, the sections were incubated overnight with Ki-67 and p-STAT3 antibodies. The sections were incubated with secondary antibodies at 37°C for 30 min, and the slides were analyzed under a light microscope. 4 Oxidative Medicine and Cellular Longevity 2.18. Statistical Analysis. Data were expressed as the mean ± standard deviation (SD). Statistical analysis was performed using the SPSS 16.0 software (SPSS, Chicago, IL, USA). Kaplan-Meier survival and log-rank tests were used for survival analysis. The correlation between THAP9-AS1 expression and SOCS3 expression in OS tissues was determined using Pearson's correlation analysis. Two-tailed Student's t -test was applied to compare the differences between two groups, and one-way analysis of variance (ANOVA) was used to compare the differences among three independent groups. p < 0:05 was considered statistically significant.

THAP9-AS1 Is Upregulated in OS Tissues and Cell Lines.
First, qRT-PCR assays were performed to evaluate the expression of THAP9-AS1 in OS cells and tissues. We found that the expression levels of THAP9-AS1 were significantly upregulated in OS cells compared with hFOB1.19 cells (p < 0:05; Figure 1(a)). The expression of THAP9-AS1 was dramatically higher in OS tissues than in nontumor tissues (1:67 ± 0:59 vs. 0:78 ± 0:56, p < 0:001; Figure 1(b)). To investigate the impact of THAP9-AS1 expression on patient survival, we divided cases into high and low groups according to the mean level of THAP9-AS1 in OS tissues. Patients with a high expression level of THAP9-AS1 tended to have a poor overall survival probability (p = 0:019; Figure 1(c)). The location of THAP9-AS1 in OS cells was investigated. As shown in Figures 1(d) and 1(e), the results of subcellular fractionation and FISH assays indicated that THAP9-AS1 might be mainly expressed in the nucleus of OS cells.      (Figure 2(a)). The expression of THAP9-AS1 was significantly upregulated in MG63 cells after transfection with THAP9-AS1-expressing vectors (p < 0:001; Figure 2(b)). We found that compared with the vector control group, the luciferase activity of cells transfected with pSOCS3-wt in the THAP9-AS1 overexpression group was reduced significantly (p < 0:01, Figure 2(c)). To investigate the binding of THAP9-AS1 to the SOCS3 promoter region, ChIP and RIP assays were performed in MG63 cells. The results of the ChIP assay showed that the methyltransferases DNMT1, DNMT3a, and DNMT3b were enriched in the promoter of SOCS3 (Figure 2(d)). The results of the RIP assay showed that THAP9-AS1 exhibited a higher conjugation rate with DNMT1, DNMT3a, and DNMT3b (Figure 2(e)). Furthermore, MSP assays validated that overexpression of THAP9-AS1 elevated the methylation of the SOCS3 promoter in both MG63 and Saos-2 cells (Figure 2(f)). Ectopic expression of THAP9-AS1 increased the number of methylated CpG sites in MG63 and Saos-2 cells compared with the vector control group (Figure 2(g)).

THAP9-AS1
Contributed to JAK2/STAT3 Signaling by Inhibiting SOCS3. Considering the inhibitory role of SOCS3 in the activation of the JAK2/STAT3 signaling pathway, the effect of THAP9-AS1 on JAK2/STAT3 signaling was investigated. We found that the protein expression of p-JAK2 and p-STAT3 (Tyr705) was induced by THAP9-AS1 but diminished by SOCS3 (Figure 4(a)). Reintroduction of SOCS3 attenuated the stimulatory effect of THAP9-AS1 on p-JAK2 and p-STAT3 (Figure 4(a)). Considering that p-STAT3 in the nucleus is essential for the transcription of its downstream target genes, we examined whether nuclear p-STAT3 accumulation was altered by THAP9-AS1 and SOCS3. The results of the IF assay demonstrated that the nuclear translocation of p-STAT3 protein was induced by THAP9-AS1 but impaired by SOCS3 (Figure 4(b)). These 3.5. The THAP9-AS1/SOCS3/JAK2/STAT3 Signaling Pathway Regulated OS Cellular Function. Cells were divided into four groups transfected with different vectors: vector control, THAP9-AS1-expressing vector, THAP9-AS1 plus SOCS3-expressing vector, and THAP9-AS1 plus AG490 (50 μM, JAK2/STAT3 signaling inhibitor). The protein expression of p-STAT3 (Tyr705) was determined to evaluate the transfection efficiency ( Figure 5(a)). We found that ectopic expression of THAP9-AS1 promoted cell proliferation, whereas reintroduction of SOCS3 exhibited an inhibitory effect on cell proliferation ( Figure 5(b)). Treatment with AG490 abolished the promoting effect of THAP9-AS1 ( Figure 5(b)). Cell apoptosis was inhibited by THAP9-AS1 and recovered in the groups treated with either SOCS3 or AG490 ( Figure 5(c)). Upregulation of THAP9-AS1 increased the number of migrated and invaded cells, which was impaired by SOCS3 (Figures 5(d) and 5(e)). Inhibition of JAK2/STAT3 signaling by treatment with AG490 significantly reversed the promoting effect of THAP9-AS1 on cell migration and invasion (Figures 5(d) and 5(e)). In addition, the protein expression of Bcl-2 and N-cadherin (a marker of epithelial-mesenchymal transition (EMT)) was elevated, and E-cadherin (a marker of EMT) was suppressed in the THAP9-AS1 groups ( Figure 5(f)). These results suggested that THAP9-AS1 contributed to cell growth and that movement might be correlated with the SOCS3/JAK2/STAT3 signaling pathway.
3.6. THAP9-AS1 Restrained Oxidative Stress through the SOCS3/JAK2/STAT3 Signaling Pathway. We monitored mitochondrial function because the role of mitochondria goes beyond their capacity to create molecular fuel and includes the generation of ROS. As shown in Figure 6(a), compared to the control group, overexpression of THAP9-AS1 notably reduced ROS generation, indicating a decline in cellular oxidative stress. Overexpression of SOCS3 or treatment with AG490 restored ROS levels ( Figure 6(a)). The mitochondrial membrane potential was observed using JC-1 staining. As shown in Figure 6(b), the OS cells in the THAP9-AS1 overexpression group exhibited high mitochondrial membrane potential, as evidenced by more red fluorescence. By comparison, the mitochondria in either SOCS3-or AG490-treated cells exhibited more green fluorescence, indicating a reduction in mitochondrial membrane potential. These data indicated that THAP9-AS1 mediated mitochondrial dysfunction in OS cells.   (Figures 7(a) and 7(b)). In mouse tumors, THAP9-AS1 was effectively overexpressed in THAP9-AS1-transfected groups

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Oxidative Medicine and Cellular Longevity (Figure 7(c)). The mRNA expression of SOCS3 was decreased in the THAP9-AS1 groups (Figure 7(d)). The results of the IHC assay validated that Ki-67, a proliferation marker accompanied by p-STAT3, was upregulated in the THAP9-AS1 group (Figure 7(e)). Moreover, we found that the overexpression of THAP9-AS1 contributed to lung metastasis of MG63 cells (Figure 7(f)).

Discussion
Accumulating evidence has proven that abnormalities in lncRNAs play critical roles in the development of cancers by affecting various signaling pathways. Our findings provide evidence demonstrating that overexpression of THAP9-AS1 leads to promoter hypermethylation of SOCS3, which further silences SOCS3 expression and activates the JAK2/STAT3 signaling pathway, thereby promoting tumorigenesis and metastasis and suppressing oxidative stress. First, we found that the expression of THAP9-AS1 was increased in OS cells and tissues. High expression of THAP9-AS1 indicated the aggressive phenotypes of tumors and the worse clinical outcome of patients. These data suggested that THAP9-AS1 may play a role in the progression of OS. THAP9-AS1 is overexpressed in PDAC tissues and positively associated with poor survival of patients [12]. High expression levels of THAP9-AS1 were also observed in gastric cancer tissues and could be induced by Helicobacter pylori infection [18]. In ESCC, upregulation of THAP9-AS1 was positively correlated with tumor size, TNM stage, lymph node metastasis, and worse prognosis [11]. According to the microarray expression profile, THAP9-AS1 was upregulated in breast cancer tissues compared with normal control tissues [10]. Thus, dysregulation of THAP9-AS1 may have important roles in a wide range of human malignancies.
The results of subcellular fractionation and FISH assays showed that THAP9-AS1 was mainly expressed in the nucleus of MG63 cells. Thus, THAP9-AS1 may regulate gene expression through an epigenetic mechanism by interacting with DNMTs. Critical DNMT family members, including DNMT1, DNMT3a, and DNMT3b, were closely associated with transcription silencing. The main function of DNMT1 is to maintain the methylation status after DNA synthesis, while DNMT3a and DNMT3b are involved in the de novo methylation of DNA [19]. As an epigenetic modification, DNA methylation is an important cellular process by which methyl groups are added to DNA molecules and leads to the initiation and progression of cancers [20]. High methylation of the SOCS3 promoter has been reported in several cancers, such as lung cancer and hepatocellular and cervical carcinomas [21][22][23]. The activation of STAT3 signaling and the inhibited expression of SOCS3  14 Oxidative Medicine and Cellular Longevity may be responsible for aggressive tumor growth in DNMT3b-positive ESCC cells [24]. Previous studies have validated that DNMT1 negatively regulates SOCS3 expression in several types of cancer cells [25][26][27]. In the present study, we found that the demethylation agent 5-aza restored SOCS3 mRNA expression to different degrees. Silencing of DNMT1, DNMT3a, and DNMT3b restored the mRNA and protein levels of SOCS3 in OS cells. These data suggested that DNA methylation may be involved in the regulation of SOCS3 in OS progression. The results of ChIP, RIP, MSP, and BSP assays validated that THAP9-AS1 might recruit DNMTs to the promoter of SOCS3 and lead to the hypermethylation status of the SOCS3 promoter region. Moreover, the mRNA levels of SOCS3 were significantly reduced in OS tissues and negatively correlated with THAP9-AS1. These data suggested that THAP9-AS1 may be an explanation for the decrease in SOCS3 in the carcinogenesis of OS. Low expression of SOCS3 was associated with TNM stage and tumor size, suggesting that SOCS3 may influence OS. We found that THAP9-AS1 contributed to the nuclear transfer of p-STAT3 and that the protein expression of p-JAK2 and p-STAT3 (Tyr705) in OS cells was reversed by SOCS3. These data revealed that THAP9-AS1 activated JAK2/STAT3 signaling by inhibiting SOCS3 in OS. MiR-19b, an increased molecule, activates the JAK2/STAT3 signaling pathway by targeting SOCS3 in OS [28]. MiR-4449 promoted cell proliferation and JAK2/STAT3 signaling activity by inhibiting SOCS3 in colorectal cancer [29]. Our data expanded the knowledge of THAP9-AS1 as a possible upstream regulator of the SOCS3/JAK2/STAT3 pathway in OS development.
Functionally, ectopic expression of THAP9-AS1 promoted cell proliferation, migration, and invasion in vitro, which could be reversed by SOCS3. In addition, inhibition of JAK2/STAT3 signaling by introducing AG490 attenuated the cancer-promoting activity of THAP9-AS1. The facilitating role of THAP9-AS1 in tumor growth and lung metastasis was also validated by animal experiments. We suggest that THAP9-AS1 may regulate gene expression and downstream pathways by interacting with DNMTs. However, THAP9-AS1 exerts its oncogenic function by sponging miR-133b and miR-484 in ESCC and PDAC, respectively [11,12]. It is possible that THAP9-AS1 may affect tumorigenesis through multiple molecular mechanisms in cancers. Furthermore, our data suggested that THAP9-AS1 decreased ROS production and increased the membrane potential of OS cells, suggesting that alteration of oxidative stress may participate in THAP9-AS1-regulated OS progression. It was demonstrated that both nuclear and mitochondrial STAT3 lead to impaired ROS generation and increased ROS scavenging activity [30,31].

Conclusions
In summary, our results revealed that THAP9-AS1 was upregulated in OS tissues and correlated with clinical stage. High THAP9-AS1 contributed to the inhibition of SOCS3 and the activation of the JAK2/STAT3 signaling pathway, thus exerting oncogenic functions in OS pathogenesis  showing that THAP9-AS1 contributed to cell proliferation and metastasis and inhibited ROS production, probably through epigenetically suppressing SOCS3, thereby activating the JAK2/STAT3 signaling pathway. * * p < 0:01; * * * p < 0:001. (Figure 7(g)). We suggest that THAP9-AS1 may serve as a novel biomarker and that THAP9-AS1/SOCS3/JAK2/-STAT3 may serve as a therapeutic strategy for the treatment of OS.

Data Availability
All data related to this paper may also be requested from the corresponding author.