LncRNA ROR1-AS1 accelerates osteosarcoma invasion and proliferation through modulating miR-504

Long non-coding RNAs (LncRNAs) play vital roles in the progression and development of tumors. However, the functional role of ROR1-AS1 in osteosarcoma has not been investigated. We found that ROR1-AS1 was upregulated in osteosarcoma tissues compared to non-tumor samples. Elevated expression of ROR1-AS1 promoted cyclin D1, PCNA and ki-67 expression and increased cell cycle and growth in MG-63 cell. Moreover, overexpression of ROR1-AS1 induced cell migration in MG-63 cell, promoting N-cadherin and vimentin expression and inhibiting E-cadherin expression. Dual-luciferase assay proved that ROR1-AS1 served as one sponge for miR-504 and ROR1-AS1 overexpression suppressed miR-504 expression in MG-63 cell. ROR1-AS1 expression was lower in osteosarcoma tissues compared to non-tumor samples. Pearson's correlation assay showed a negative correlation between miR-504 and ROR1-AS1 expression. MiR-504 overexpression partly abrogated ROR1-AS1-induced effects on osteosarcoma cell migration and proliferation. These data implied that ROR1-AS1 played as an oncogene and might be a new treatment target for osteosarcoma.


INTRODUCTION
Osteosarcoma was a common type of malignant bone tumor among young adults and teens and originated from specimen of osteoid bon [1][2][3][4][5]. Osteosarcoma is characterized by to high metastasis rate, Although several treatment advancements including postoperative chemotherapy and surgical resection have been attained, overall survival (OS) rate of osteosarcoma cases remains unsatisfactory due to recurrence and metastasis [6][7][8][9][10]. Until now, detailed role and mechanism of metastasis and oncogenesis remained unclear in osteosarcoma [11,12]. Thus, it is vital to investigate the modulatory mechanism of osteosarcoma and seek new treatment strategies for this disease.

MiR-504 expression level in osteosarcoma tissues
RT-qPCR assay was carried out for measuring miR-504 level in 30 samples. The detailed expression of miR-504 was shown in Figure 5A and 5B and miR-504 level was downregulated in 21 cases (27/35, 77.1%) compared to non-tumor samples. Furthermore, ROR1-AS1 expression was lower in osteosarcoma tissues compared to non-tumor samples ( Figure 5C). Pearson's correlation assay showed a negative correlation between miR-504 and ROR1-AS1 expression ( Figure 5D).

DISCUSSION
Numerous references have indicated that lncRNAs play vital roles in several biological processes such as tumor initiation and growth [23,35,36]. We identified that ROR1-AS1 expression was higher in osteosarcoma tissues compared to non-tumor samples. Elevated expression of ROR1-AS1 promoted cyclin D1, PCNA and ki-67 expression and increased cell cycle and growth in MG-63 cell. Moreover, overexpression of ROR1-AS1 induced cell migration in MG-63 cell, promoting N-cadherin and vimentin expression and inhibiting E-cadherin expression. Dual-luciferase assay proved that ROR1-AS1 functioned as a sponge for miR-504 and ROR1-AS1 overexpression suppressed miR-504 expression in MG-63 cell. ROR1-AS1 expression was lower in osteosarcoma tissues compared to nontumor samples. Pearson's correlation assay showed a negative correlation between miR-504 and ROR1-AS1 expression. MiR-504 overexpression partly abrogated ROR1-AS1-induced effects on osteosarcoma cell migration and proliferation. These data implied that ROR1-AS1 played as an oncogene and might be a new treatment target for osteosarcoma. ROR1-AS1 was located in the 1p31.3 and was one novel identified lncRNA in lymphoma [30]. Growing evidences have showed that ROR1-AS1 acts as AGING oncogenes in some tumors including nasopharyngeal carcinoma, bladder cancer and colorectal cancer [32][33][34]. ROR1-AS1 expression was upregulated in nasopharyngeal carcinoma samples and ROR1-AS1 knockdown inhibited cell invasion and migration and EMT progression in nasopharyngeal carcinoma cell via regulating miR-375 [33]. Chen et al [34]. found that ROR1-AS1 was overexpressed in bladder tumor samples and associated with lymph node metastasis, advanced stage and histological grade. ROR1-AS1 knockdown decreased bladder tumor cell migration and proliferation. Wang et al. [32] indicated that ROR1-AS1 expression was upregulated in colorectal cancer samples and ROR1-AS1 downregulation suppressed cell invasion and migration in colorectal cancer cell through regulating miR-375. However, the functional role of ROR1-AS1 in osteosarcoma remains uninvestigated so far. We observed that ROR1-AS1 expression was higher in osteosarcoma tissues compared to non-tumor samples and ROR1-AS1 overexpression induced osteosarcoma cell migration and proliferation.
In summary, we identified that ROR1-AS1 expression was overexpressed in osteosarcoma tissues and cell and ROR1-AS1 overexpression promoted cell migration, EMT progression and migration via modulating miR-504. Thus, ROR1-AS1 played as one oncogene and might be a new treatment target for osteosarcoma.

Specimens, cell culture and transfection
Human osteosarcoma specimens along with normal adjacent specimens were obtained at our hospital. This research was conducted according to Declaration of Helsinki and approved with Nanyang hospital. Each case provided consent signed forms. Cell lines of osteosarcoma (SAOS-2, U2OS, MG-63 and HOS) and osteoblast cell line (hFOB1. 19) were utilized in our reference. pcDNA-ROR1-AS1, miR-504 mimic, pcDNA-control and scramble were getting from GenePharma (Shanghai, China). Cell transfections were performed by Lipofectamin3000 (Invitrogen Inc.)

Cell growth and migration assay
CCK-8 analysis was utilized to detect cell growth. Transfected osteosarcoma cells were cultured in the plate of 96-well and 10 μL CCK-8 kit was cultivated with cells for 2 hours. Absorbance at the 450 nm was calculated by microplate reader. Wound healing analysis was utilized to study cell migration. Cells were plated in dish of 6-well and continued to confluence. Pipette tip was used to scratch cell wound and cells were plated in serum-free medium. Relative wound distance was recorded by microscope (Olympus).

Statistical analysis
Statistical assay was carried out by SPSS (19.0, SPSS Inc, USA). Data were presented as mean ±standard deviation. Student's t-test was utilized to determine significant differences. The value of < 0.5 was supposed to be significant.

CONFLICTS OF INTEREST
These authors declare no conflicts of interest.

AUTHOR CONTRIBUTIONS
Xiangkun Wu, Lihua Yan, Yongxi Liu, Lilin Shang conceived and designed the project. Xiangkun Wu and Lihua Yan performed experiments and/or data acquisition and analyses; Yongxi Liu contributed technical/reagents materials, analytic tools; Xiangkun Wu, Lihua Yan and Yongxi Liu prepared, wrote, AGING and/or revision the manuscript. All authors discussed the results and commented on the manuscript.