lncRNA DARS-AS1 Promoted Osteosarcoma Progression through Regulating miR-532-3p/CCR7

Background lncRNAs have been indicated to involve in cell invasion, proliferation, and metastasis. However, function of DARS-AS1 in osteosarcoma remains poorly explored. Methods DARS-AS1 and miR-532-3p level were measured using qRT-PCR. CCK-8 assay and cell invasion assay were done to study cell functions. Luciferase reporter assay was performed to study the mechanism about DARS-AS1 and miR-532-3p. Results We firstly showed that DARS-AS1 expression is upregulated in 73.5% (25/34) of cases with osteosarcoma. Moreover, DARS-AS1 expression is overexpressed in osteosarcoma specimens than in nontumor samples. The DARS-AS1 is overexpressed in the osteosarcoma cell lines (Saos-2, SOSP-9607, U2OS, and MG-63) compared to hFOB. Overexpression of DARS-AS1 promotes cell growth and invasion in MG-63 osteosarcoma cell. DARS-AS1 plays as one sponge for miR-532-3p in osteosarcoma cell, and miR-532-3p overexpression inhibits luciferase activity of DARS-AS1-WT, not DARS-AS1-MUT in MG-63 cell. Ectopic expression of DARS-AS1 inhibits miR-532-3p expression in MG-63 cell. Furthermore, miR-532-3p expression is downregulated in osteosarcoma specimens compared to in paired nontumor samples. MiR-532-3p expression is downregulated in osteosarcoma cell lines compared to hFOB. MiR-532-3p expression is negatively associated with DARS-AS1 expression in osteosarcoma specimens. miR-532-3p directly regulates CCR7 expression in osteosarcoma cell. Elevated DARS-AS1 expression enhances cell growth and invasion via regulating CCR7. Conclusions These data firstly suggested that DARS-AS1 exerted as one oncogene in osteosarcoma partly via regulating miR-532-3p/CCR7.


Background
Osteosarcoma is a primary bone malignancy that influences growing bones of adolescents and children and is interrelated with high morbidity [1][2][3]. The development of several therapeutic methods for osteosarcoma such as radiotherapy, multiagent chemotherapy, and precise tumor excision has ameliorated the prognosis of osteosarcoma [4][5][6]. However, the five-year surgical rate of these patients diagnosed with advanced stage is still discontent [7][8][9]. Thus, it is urgent to find novel biomarkers and treatment targets for osteosarcoma cases.

Cell Proliferation and Invasion
Assay. For cell proliferation, cell was kept into the 96-well dish at 5 × 10 3 cells/well. The cell growth rate was analyzed using CCK-8 (DOJINDO, Japan) following manufacturer's protocol at the different time points. The absorbance at 450 nM was determined on the microtiter reader. For cell invasion, Bio-Coat Matrigel chambers (BD Biosciences, Germany) was used. For cell invasion assay, cells were seeded on the top chamber (Matrigel coated filter) in the serum-free medium, and FBS (10%) was conducted as a chemoattractant. After incubation for 48 hours, the cell that invaded to lower side was fixed and counted.
2.5. Luciferase Reporter Assay. Cell was cotransfected pMIR vector containing the diverse mutant or wild type DARS-AS1 and mutant or wild type CCR7, along with pRL-TK control plasmid and miR-532-3p mimic or scramble control by using Lipofectamine2000 (Invitrogen, USA). After 2 days, cell was harvested and then analyzed with the Dual Lucifer-ase Assay kit (Promega, USA) following manufacturer's protocol.
2.6. Statistical Analysis. Data are indicated as means + SD (Standard Deviation) based on 3 independent experiments and determined by using SPSS version 12.0 software (SPSS, Chicago, USA). Statistical significance was regarded as P value < 0.05. The significant difference was analyzed by one-way analysis of variance or Student's t tests.

Discussion.
Our study identified that DARS-AS1 acted as one oncogenic lncRNA in the development of osteosarcoma. We firstly revealed that DARS-AS1 expression was higher in osteosarcoma specimens than in paired nontumor samples. DARS-AS1 promoted cell growth and invasion in MG-63 osteosarcoma cell. We found that DARS-AS1 played as a sponge for miR-532-3p in osteosarcoma cell, and ectopic expression of DARS-AS1 inhibited miR-532-3p level in MG-63 cell. Furthermore, miR-532-3p expression was lower in osteosarcoma specimens than in nontumor samples and miR-532-3p expression was negatively associated with DARS-AS1 expression in osteosarcoma specimens. miR-532-3p directly regulated CCR7 expression in osteosarcoma cell. Elevated expression of DARS-AS1 enhanced cell growth and invasion via regulated CCR7. These data suggested that DARS-AS1 exerted as one oncogene in osteosarcoma partly via regulating miR-532-3p/CCR7. Studies revealed that DARS-AS1 exerted an oncogenic role in several human tumors such as thyroid cancer, lung cancer, myeloma, and ovarian cancer [25][26][27][28]. For example, Zheng et al. [27] revealed that DARS-AS1 expression was increased in thyroid tumor specimens and was associated with poor prognosis, distant metastasis, and pathological stage, and DARS-AS1 facilitated thyroid tumor cell migration and proliferation via regulating miR-129. Liu et al.    Disease Markers [28] found that DARS-AS1 induced nonsmall cell lung tumor progression through modulating miR-532-3p. Yan [26] showed that DARS-AS1 was upregulated via HIF-1 in myeloma. DARS-AS1 induced myeloma cell tumorigenesis and survival via binding RBM39. Huang et al. [25] indicated that expression of DARS-AS1 was upregulated in ovarian tumor specimens, and silenced DARS-AS1 expression suppressed cell invasion, migration, and proliferation via modulating miR-532-3p. Its role in osteosarcoma remains poorly explored. We firstly studied the expression level of DARS-AS1 in osteosarcoma specimens and paired nontumor samples. Our data indicated that DARS-AS1 expression was upregulated in 73.5% (25/34) of cases with osteosarcoma. Moreover, DARS-AS1 expression was higher in osteosarcoma specimens than in paired nontumor samples. DARS-AS1 promoted cell growth and invasion in MG-63 osteosarcoma cell. These results suggested that DARS-AS1 acted as one oncogenic role in the development of osteosarcoma. Recent references have suggested that lncRNAs played roles in a lot of tumors via modulating miRNAs expression [16,[29][30][31]. For instance, lncRNA HOXA-AS2 suppressed osteosarcoma cell invasion, viability, and migration via sponging miR-124-3p [32]. Li et al. [33] indicated that lncRNA NR2F1-AS1 promoted osteosarcoma progression through sponging miR-483-3p. lncRNA SND1-IT1 promoted osteosarcoma migration and proliferation through regulating miRNA-665 expression [34]. lncRNA SPRY4-IT1 induced osteosarcoma progression through sponging miR-101 [35]. Moreover, lncRNA DARS-AS1 promoted ovarian cancer cell metastasis and growth via sponging miR-532-3p [25]. We also observed that DARS-AS1 has potential binding sites of miR-532-3p in osteosarcoma. Previous study demonstrated that miR-532-3p expression was downregulated in the osteosarcoma tissues [36]. We also found that miR-532-3p expression was lower in osteosarcoma specimens than in paired nontumor samples. Moreover, the data of Pearson's correlation assay indicated that miR-532-3p expression was negatively associated with DARS-AS1 expression in osteosarcoma specimens. miR-532-3p directly regulated CCR7 expression in osteosarcoma cell. Previous study demonstrated that miR-532-3p inhibited TSCC progression through regulating CCR7 and it suggested that CCR7 might play important roles in the development of osteosarcoma [37]. CCR7 are involved in tumor migration and metastasis [38]. We firstly showed that elevated expression of DARS-AS1 enhanced cell growth and

Data Availability
The authors can make data available on request through an email to the corresponding author, enrxiaoping@126.com, Prof. Dr. Ren.

Ethical Approval
This study was performed with the approval of Clinical Ethics Committee of the Second Affiliated Hospital of Harbin Medical University.

Consent
Each patient has written an informed consent.

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