LncRNA LINC00662 Exerts an Oncogenic Effect on Osteosarcoma by the miR-16-5p/ITPR1 Axis

Background Osteosarcoma (OS) is one of the most malignant bone tumors and has a high metastatic rate. Increasing research has demonstrated the vital roles of long noncoding RNAs (lncRNAs) in human cancers, including OS. LncRNA LINC00662 has been revealed to act as an oncogene involved in multiple tumor progression. This study aimed to investigate the expression pattern, function, and regulatory mechanism of LINC00662 in OS. Methods Patients who underwent OS surgery were involved in this study. Experiments including RT-qPCR, MTT, western blot, FISH, RNA pull-down, luciferase reporter, colony formation, transwell invasion and migration, and sphere formation assay were performed to investigate the regulatory role of LINC00662 in OS. Results In the present study, our findings demonstrated the upregulation of LINC00662 expression in OS tissues and cells, and high expression of LINC00662 predicted a poor clinical prognosis of patients' iNOS. Through a series of in vivo assays, LINC00662 knockdown suppressed OS cell proliferation, invasion, migration, and stemness property maintenance. Further mechanistical investigations indicated that LINC00662 functioned as a competing endogenous RNA (ceRNA) for sponging microRNA-16-5p (miR-16-5p) to upregulate the expression of IP receptor type 1 (ITPR1) in OS cells. Restoration assays validated the involvement of ITPR1 in LINC00662-mediated regulation of cell functions in OS. Conclusion LINC00662 exerts oncogenic functions in OS by targeting the miR-16-5p/ITPR1 axis.


Introduction
OS is a common malignant bone tumor with rapid metastasis, affecting a multitude of people, frequently during adolescence [1][2][3]. High genetic instability and extreme genome are two main factors contributing to the occurrence and development of OS [4]. Despite significant advances in clinical therapies including surgery, chemotherapy, and radiotherapy, a long-term survival rate for patients with OS remains unsatisfying [5]. Notably, the 5-year survival rate decreases from 77.3% to 33.9% when metastasis occurs in OS patients [6]. Considering that the pathogenesis of OS is quite complex [7], it is urgent to investigate the molecular mechanisms underlying OS progression for improvement of the diagnosis and treatment.
Long noncoding RNAs (lncRNAs) are a kind of molecules longer than 200 nucleotides and lack protein-coding ability [8], playing crucial roles in various biological processes via different mechanisms [9]. Accumulating evidence has revealed the regulatory roles of lncRNAs in tumor progression. For example, lncRNA ASB16-AS1 mediates the proliferation and invasion of hepatocellular carcinoma cells by regulating the miR-1827/FZD4 axis [10]. LncRNA CCAT1 promotes colorectal cancer cell proliferation in vitro and in vivo by upregulating TUSC3 [11]. Multiple lncRNAs have been discovered to participate in OS progression. For example, lncRNA NR2F1-AS1 is overexpressed in OS, and its knockdown suppresses cell proliferation by inducing cell cycle arrest and promotes cell apoptosis [12]. LncRNA SND1-IT1 sponges miR-665 to upregulate POU2F1 expression, thereby facilitating OS cell proliferation and migration [13]. LncRNA LINC00662 has also been reported to be a key regulator of biological behaviors in many cancers. LINC00662 is upregulated in hepatocellular carcinoma and contributes to cell proliferation and invasion through activating Wnt/β-catenin signaling pathway [14]. LINC00662 acts as a miR-34a sponge to promote the tumorigenesis of prostate cancer [15]. LINC00662 has been considered a valuable prognostic biomarker in colon cancer [16]. Since the oncogenic effect of LINC00662 has been well clarified in other cancers, we hypothesized that LINC00662 might play a role in OS. Moreover, LINC00662 has been reported to function as a competing endogenous RNA (ceRNA) by sponging miRNAs in cancers. We thus investigated whether LINC00662 could exert ceRNA functions in OS.
In this research, we aimed to investigate the expression pattern of LINC00662 in OS clinical samples and the function of LINC00662 in OS cell proliferation, migration, invasion, and stemness characteristics, as well as the potential regulatory mechanisms of LINC00662 in OS, which might provide a novel insight for OS diagnosis and treatment.

Clinical Tissues.
OS tissues and adjacent normal tissues from 51 patients who underwent surgical resection were collected from Foshan Hospital of Traditional Chinese Medicine (Guangdong, China) from January 2014 to December 2017. e enrolled patients were within the age of 26-65, with an average age of 45.87 ± 11.02, and they were at different TNM stages (I-VI). All patients were clinically and pathologically diagnosed. None of patients had received any antitumor therapy including radiotherapy and chemotherapy before the operation. e collected tissues were immediately frozen with liquid nitrogen and stored at −80°C until use. All patients signed written informed consent. is study was approved by the Ethics Committee of Foshan Hospital of Traditional Chinese Medicine (Guangdong, China). Follow-up of the patients via phone or return visit was performed every 3 months during the first year after surgery until December 2019 with the observation of overall survival rate of these patients.
2.6. Colony Formation Assay. After transfection, U2OS or SAOS-2 cells were seeded in 6-well plates (2 × 10 5 cells/well). e medium was replaced every 3 days. Cells were cultured for 14 days at 37°C before the medium was removed. en, cells were washed with phosphate buffer saline (PBS). Afterwards, the colonies were fixed in 10% paraformaldehyde for 10 min and stained with 0.1% crystal violet solution 2 Journal of Oncology (Beyotime, shanghai, China) for 10 min. e number of colonies was calculated with an inverted microscope.

Transwell Invasion and Migration Assays.
Migratory and invasive ability of OS cells were assessed using a 24-well transwell chamber (8 μm; Costar, Boston, MA, USA). For invasion detection, after transfection, 2 × 10 5 U2OS or SAOS-2 cells in serum-free medium were added to the upper chamber precoated with Matrigel (BD Biosciences, CA, USA), while the lower chamber was filled with 600 μL RPMI-1640 medium containing 10% FBS. After 36 h of transfection, the invaded cells were fixed with 70% methanol and stained with 0.1% crystal violet (Sigma-Aldrich, USA). For migration detection, the upper chamber was not precoated with Matrigel.

Western Blot Analysis.
e lysates of U2OS or SAOS-2 cells were obtained using an RIPA lysis buffer (Beyotime), and protein quantification was performed using a BCA protein assay kit (Pierce, Rockford, USA). Total proteins were separated by 12% SDS-PAGE gel and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, MA, USA), which were blocked with 5% skimmed milk for 1 h at room temperature. en, primary antibodies (all from Boster, Wuhan, China) were incubated with the membranes overnight at 4°C. Afterwards, the membranes were washed with TBST three times and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Cell Signaling Technology) for 2 h as per the supplier's protocols.

RNA Fluorescence In Situ Hybridization (FISH).
e LINC00662-specific RNA FISH probe was commercially designed and synthesized by RiboBio (Guangzhou, China). A FISH Kit (RiboBio) was used to determine the probe signals. In brief, U2OS, SAOS-2, or HFOB 1.19 cells were fixed in 4% formalin for 15 min. e cells were hybridized again in hybridization solution at 37°C for 30 min after prehybridization in PBS. Next, DAPI staining (Beyotime) was performed to counterstain the cell nuclei. e images of staining were captured using a fluorescence microscope (Leica, Germany).

RNA Pull-Down
Assay. LINC00662 biotin probe and LINC00662 no-biotin probe, as well as miR-16-5p biotin probe and miR-16-5p no-biotin probe, were synthesized by Sangon (Shanghai, China). e cell protein extracts from U2OS or SAOS-2 were collected for incubation with the RNA or NC probes overnight. en, the lysate was added with streptavidin agarose beads (Life Technologies) and incubated for another 1 h at room temperature. Next, these beads were boiled in SDS buffer and purified by RNeasy Mini Kit (Qiagen, USA). e purified RNA was detected by RT-qPCR.
2.13. Statistical Analysis. All data are presented as the mean ± standard deviation (SD). Statistical analysis was performed with SPSS 19.0 software (SPSS, Chicago, USA). Statistical differences between two groups were analyzed using student's t-test and those among groups using oneway analysis of variance (ANOVA). P < 0.05 was considered statistically significant. All experiments were conducted more than three times.

LINC00662 is Upregulated in OS Tissues and Cells.
Design of the present study is shown in Supplementary  Figure 1. To determine the role of LINC00662 in OS, we first detected the expression of LINC00662 in 51 paired OS tissues and adjacent normal tissues using RT-qPCR. e results showed that LINC00662 expression was higher in OS tissues than in normal tissues ( * * P < 0.01; Figure 1(a)). Subsequently, the expression of LINC00662 in OS cells was measured. As expected, LINC00662 expression was significantly upregulated in OS cell lines (U2OS, SAOS-2, 143B, and MG63) compared with the human osteoblast cell line HFOB 1.19 ( * * P < 0.01; Figure 1(b)). U2OS cells exhibited the highest level of LINC00662, followed by SAOS-2 cells.
erefore, U2OS and SAOS-2 cells were used for further assays. To investigate whether LINC00662 expression is correlated to the clinicopathological characters of patients, 51 patients were divided into a low-LINC00662-expression group (n � 25) and a high-LINC00662-expression group (n � 26) according to the median LINC00662 expression. As shown in Table 1, the high-LINC00662 level was significantly related to distant metastasis (P < 0.001), TNM stage (P < 0.001), and tumor size (P � 0.007). To further analyze whether LINC00662 could act as a prognostic biomarker for Journal of Oncology 3 OS, a clinical follow-up study was conducted to determine the overall survival of patients. Kaplan-Meier analysis indicated that the patients in the high-LINC00662-expression group had shorter overall survival compared with those in the low-LINC00662-expression group (Figure 1(c)). In univariate analysis, distant metastasis, TNM stage, tumor size, and increased LINC00662 expression were potential risk factors of shorter overall survival (P � 0.009; P � 0.040; P � 0.015; P � 0.023). After adjustment for confounding factors, the multivariate analysis indicated that metastasis   Journal of Oncology and increased LINC00662 expression (P � 0.012; P � 0.013) were independently associated with shorter overall survival in OS patients (Table 2).

LINC00662 Knockdown Inhibits Malignant Phenotypes of OS Cells.
Since the high expression level of LINC00662 was confirmed, we then intended to explore the role of LINC00662 in OS. RT-qPCR analysis was utilized to check the transfection efficiency of sh-LINC00662 and showed that LINC00662 expression was effectively knocked down in U2OS and SAOS-2 cells after transfection ( * * P < 0.01; Figure 2(a)). e results of MTTand colony formation assays revealed that cell viability and proliferation were significantly inhibited in U2OS and SAOS-2 cells after silencing LINC00662 ( * * P < 0.01; Figures 2(b) and 2(c)). Transwell assays confirmed that inhibition of LINC00662 reduced the invasive and migratory abilities of OS cells ( * * P < 0.01; Figures 2(d) and 2(e)). Additionally, the effect of LINC00662 downregulation on OS stem cell properties was investigated.

ITPR1 Reverses the Regulatory Effect of LINC00662 Knockdown in OS Cells.
To further test whether ITPR1 expression is responsible for the function of LINC00662, the expression of ITPR1 at mRNA and protein levels was restored using pcDNA3.1/ITPR1 in U2OS cells with LINC00662 silencing ( * * P < 0.01; Figure 5

Discussion
In recent years, increasing evidence has revealed that lncRNAs could exert important regulatory functions on the occurrence and development of various cancers, including OS [9-11, 13, 18], demonstrating the potential research value of lncRNAs in the molecular mechanisms underlying cancer progression. e biological roles of LINC00662 have been studied in a few types of cancers. For example, LINC00662 facilitates cell migration, invasion, and stemness Journal of Oncology     maintenance via interacting with LIN28 in lung cancer [19].
Inhibition of LINC00662 reduces cell proliferative, migratory, and invasive abilities of in oral squamous cell carcinoma [20]. LINC00662 mediates the malignant phenotypes of colorectal cancer cells by the miR-145/c-myc axis [21]. In our research, we demonstrated that LINC00662 was significantly upregulated in OS tissues and cell lines and was negatively related to the overall survival of patients with OS. en, we found that depletion of LINC00662 significantly inhibited OS cell proliferation, migration, invasion, and stem cell properties in vitro. ese findings exhibited the carcinogenic role of LINC00662 in OS, which is consistent with the previous studies.
ere has been increasing attention drawn to the theory of ceRNA network; that is, lncRNAs function as sponges for miRNAs to modulate the expression of the target genes [22]. Accumulating research has indicated that lncRNAs could exert ceRNA functions in cancers. For example, lncRNA LINC00460 enhances the progression of head and neck squamous cell carcinoma through sponging miR-612 and upregulating AKT2 [23]. LncRNA SPRY4-IT1 binds to miR-6882-3p by competing with TCF7L2 to affect the cell stemness of breast cancer [24]. LncRNA CHL1-AS1 increases the abilities of cell proliferation and migration via sponging miR-6076 to upregulate CHL1 expression in endometrial cancer [25]. In addition, LINC00662 has been reported to serve as a ceRNA in some cancers [26,27]. In this study, we examined the subcellular localization of LINC00662 in OS cells and found that LINC00662 was mostly distributed in the cytoplasm, suggesting the possibility that LINC00662 might be involved in ceRNA network by sponging miRNAs. MicroRNAs, a group of small ncRNA molecules with 20-24 nucleotides, can prevent the translation or degradation of target mRNAs at the posttranscriptional level [28]. rough online tools, miR-16-5p was predicted to bear the complementary base pairing with LINC00662. In this study, LINC00662 was validated to bind to miR-16-5p. Previous investigations have shown that miR-16-5p is differentially expressed and plays a key role in the progression of numerous cancers, such as breast cancer [29], cervical cancer [30], and hepatocellular carcinoma [31]. In the present study, miR-16-5p was expressed at a low level in OS tissues and cells and negatively related to LINC00662 in terms of expression level. Conclusively, LINC00662 functions as a molecular sponge for miR-16-5p in OS cells.
To further support the ceRNA pattern in OS, the target genes of miR-16-5p were investigated. Based on the bioinformatics tools, ITPR1 (inositol 1,4,5-trisphosphate receptor, type 1) was identified. ITPR1 has been reported to be involved in Ai-lncRNA EGOT-mediated paclitaxel resistance in human cancers [32]. ITPR1 serves as a direct target of EPAS1 and an autophagy regulator to protect renal carcinoma cells against NK-mediated killing [33]. In this study, ITPR1 was verified to be a direct target of miR-16-5p. Meanwhile, ITPR1 was highly expressed in OS tissues and cells. In addition, rescue assays confirmed that overexpression of ITPR1 could reverse the LINC00662 silencinginduced inhibitive effect on OS cell functions.
In conclusion, this study investigated the biological role and regulatory mechanisms of LINC00662 in OS. Our data support evidence that LINC00662 exerts ceRNA function by sponging miR-16-5p to upregulate ITPR1, thereby mediating the malignant phenotypes of OS cells. Our findings might provide an instructive insight for the future exploration of OS treatment.

Data Availability
e datasets used or analyzed during the current study are available from the corresponding author on reasonable request.

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