LncRNA BCRT1 facilitates osteosarcoma progression via regulating miR-1303/FGF7 axis

Growing studies noted that lncRNA was closely related with the initiation and progression of tumors. However, the role of BCRT1 in the progression of osteosarcoma remains unknown. We noted that BCRT1 is significantly upregulated in osteosarcoma specimens and cells. Elevated expression of BCRT1 promotes cell growth and cell cycle in osteosarcoma cell. Moreover, BCRT1 induces EMT and secretion of inflammatory mediators in osteosarcoma cell. We illustrated that elevated expression of BCRT1 decreases miR-1303 expression in MG-63 cell. The expression of miR-1303 is lower in osteosarcoma specimens than in non-tumor specimens. There is an inverse interrelation between miR-1303 levels and BCRT1 levels in osteosarcoma specimens. Furthermore, we identified FGF7 is one direct target gene of miR-1303 in osteosarcoma cell. Ectopic expression of miR-1303 suppresses FGF7 expression and elevated expression of BCRT1 enhanced FGF7 expression in MG-63 cell. Finally, we illustrated that BCRT1 induces osteosarcoma cell cycle and proliferation and promotes EMT progression and inflammatory mediators secretion via modulating FGF7 expression. Our study suggested that BCRT1 acts as one oncogene in osteosarcoma progression.


INTRODUCTION
Osteosarcoma is the 8 th leading tumor with approximately incidence of 4.4 per one million. Osteosarcoma mostly occurs in adolescents and children, contributing to about 5% of all childhood malignancies and about 9% of tumor-correlated deaths in children [1][2][3][4]. Clinical results illustrated that this cancer has an uncertain prognosis, even with comprehensive therapies including chemotherapy and amputation [3,5,6]. Extensive cancer-associated specific mediators and signaling pathways have been identified in osteosarcoma prognosis, pathogenesis and progression [7][8][9][10]. However, the molecular mechanisms about these specific mediators and signaling pathways are still largely unknown in osteosarcoma carcinogenesis. Thus, it is important to study molecular mechanisms about osteosarcoma pathogenesis.

BCRT1 is significantly upregulated in osteosarcoma specimens
The level of BCRT1 was determined in 40 osteosarcoma specimens using RT-qPCR analysis and data were shown in Figure 1A, 1B. The expression of BCRT1 is higher in osteosarcoma specimens than in non-tumor specimens ( Figure 1C).

MiR-1303 is significantly downregulated in osteosarcoma specimens
The level of miR-1303 was measured in 40 osteosarcoma specimens using RT-qPCR method and data were shown in Figure 2A, 2B. The expression of miR-1303 is lower in osteosarcoma specimens than in non-tumor specimens ( Figure 2C). As indicated in Figure 2D, Spearman's correlation assay shows an inverse interrelation between miR-1303 levels and BCRT1 levels.

BCRT1 induces EMT progression and secretion of inflammatory mediators in osteosarcoma cell
Overexpression of BCRT1 inhibits epithelial marker Ecadherin expression in MG-63 cell ( Figure 4A). Moreover, elevated expression of BCRT1 increases the expression of N-cadherin ( Figure 4B) and vimentin ( Figure 4C) in MG-63 cell, which were the mesenchymal markers. Elevated expression of BCRT1 induces secretion of several inflammatory mediators, including IL-1β ( Figure 4D), TGFβ ( Figure 4E) and IL-6 ( Figure 4F).

FGF7 induces osteosarcoma cell cycle and proliferation, EMT progression and secretion of inflammatory mediators
The level of FGF7 was overexpressed in MG-63 cell after treatment with pcDNA-FGF7 by qRT-PCR  AGING ( Figure 8A). Ectopic expression of FGF7 increased cell growth ( Figure 8B) and ki-67 ( Figure 8C) expression in the MG-63 cell. Overexpression of FGF7 increased cell cycle in MG-63 cell ( Figure 8E). Elevated expression of FGF7 suppressed E-cadherin expression and increased the expression of N-cadherin and vimentin in MG-63 cell ( Figure 8F). Ectopic expression of FGF7 induces secretion of several inflammatory mediators, including  AGING IL-1β ( Figure 8F), IL-6 ( Figure 8G) and TGFβ ( Figure 8H).

DISCUSSION
Recent studies have illustrated that lncRNAs participate in the progression of various tumors, including osteosarcoma. For instance, Liu et al [29]. noted that PGM5-AS1 induces EMT progression, metastasis and invasion of osteosarcoma cells through impairing miR-140-5p-regulated FBN1 inhibition. Liu et al [30]. showed that FOSL2 and OIP5-AS1 is overexpressed in cisplatin resistant osteosarcoma cells and tissues. Knockdown of OIP5-AS1 inhibits osteosarcoma growth and induces cisplatin sensitivity of osteosarcoma. Shen et al [31]. indicated that lncARSR confers resistance to  AGING drug Adriamycin and induced progression of osteosarcoma. Furthermore, Liang et al [28]. noted that BCRT1 is overexpressed and is associated with poor prognosis in breast tumor. Knockdown of BCRT1 inhibits tumor metastasis and growth. However, the role of BCRT1 in the progression of osteosarcoma remains unknown. In present research, we noted that BCRT1 is significantly upregulated in osteosarcoma specimens and cells. Elevated expression of BCRT1 promotes cell growth and cell cycle in osteosarcoma cell. We also noted that overexpression BCRT1 increased ki-67, cyclin D1 and CKD2 expression due to these genes were the important proliferation markers. Moreover, BCRT1 induces EMT progression and secretion of inflammatory mediators in osteosarcoma cell.
Growing literatures have illustrated that lncRNAs serve as miRNA sponges or decoys to influence their expression. For example, Wang et al [32]. found that RHPN1-AS1 acts as an oncogene in osteosarcoma via sponging miR-506/SNAI2 Expression. LncRNA LINC01419 was noted to regulate PDRG1/miR-519a-3p axis to induced osteosarcoma cell progression. Chen et al [33]. illustrated that knockdown of LINC00313 suppresses metastasis and tumorigenesis in osteosarcoma via modulating miR-342-3p/FOSL2 axis. Zhang et al [34]. showed that DSCAM-AS1 induces USP47 expression via sponging miR-101-3p to promote progression of osteosarcoma. Wang et al [35]. noted that lncRNA OR3A4 modulated osteosarcoma cells growth through regulating miR-1207-5p/G6PD axis. Recently, Liang et al [28]. showed that knockdown of BCRT1 inhibits tumor metastasis and growth through regulating miR-1303/PTBP3 axis. We also illustrated that elevated expression of BCRT1 decreases miR-1303 expression in MG-63 cell. The expression of miR-1303 is lower in osteosarcoma specimens than in non-tumor specimens. There is an inverse interrelation between miR-1303 level and BCRT1 level in osteosarcoma specimens. Furthermore, we identified FGF7 is one direct target gene of miR-1303 in osteosarcoma cell. Ectopic expression of miR-1303 suppresses FGF7 expression and elevated expression of BCRT1 enhances FGF7 expression in MG-63 cell. Finally, we illustrated that BCRT1 induces osteosarcoma cell cycle and proliferation and promotes EMT progression and inflammatory mediators secretion via modulating FGF7 expression. Overexpression of FGF7 induced osteosarcoma cell cycle and proliferation, EMT progression and secretion of inflammatory mediators.
Taken together, present study illustrated that BCRT1 is significantly upregulated in osteosarcoma specimens and BCRT1 induces osteosarcoma cell cycle and proliferation and promotes EMT progression and secretion of inflammatory mediators via modulating FGF7 expression. It suggested that BCRT1 acts as one oncogene in osteosarcoma progression.

Tissue specimens and cell culture and transfection
Osteosarcoma specimens and adjacent specimens were collected from cases undergoing surgery at our department and all cases gave informed consent. The biopsies were kept in liquid nitrogen until RNA extraction. Osteosarcoma cells (U2OS, SAOS-2, MG-63 and HOS) and hFOB1.19 cell was cultured in the RPMI-1640 medium supplemented by penicillin and FBS. miR-NC mimic and miR-1303 mimic, pcDNA-BCRT1 and pcDNA-control, siRNA FGF7 and control vectors, pcDNA-FGF7 and pcDNA-control vectors were obtained from GenePharma Company (Shanghai, China). Cell transfection was carried out using Lipofectamin3000 (Invitrogen Inc.)

Cell growth and cell cycle and colony formation
The viability of osteosarcoma cell cultured in the 96well dish was detected by CCK-8 analysis reagent (Dojindo, Japan). After treatment, 10 μL CCK-8 kit was added into each well for 3 hours. Absorbance at the 450 nm was read at plate reader. Cell stages of cycle were evaluated using flow cytometry. Cells were fixed in the ethanol (70%, ice) and treated with 10 mg/mL RNase A. Cell was stained with propidium iodide and were analyzed by FACStar sorter. For cell colony formation, cells were cultured in the 96-well and cells continued to culture for 7 days. Colonies were stained with the crystal violet and pictured.

Dual-luciferase reporter gene assay
The mut and WT reporter vectors of BCRT1 and FGF7 (mut-BCRT1 or FGF7 and WT-BCRT1 or FGF7) were synthetized from GenePharma Company (Shanghai, China). These NC mimics and miR-1303 mimics were co-transfected with the mut-BCRT1 or FGF7 and WT-BCRT1 or FGF7 into MG-63 cell. After 48 hours luciferase values were studied at luciferase reporter kit (Promega Corporation, USA) following to protocol.

Statistical analysis
All data were noted as mean ± SD (standard deviation). Student's t-test was utilized to compare the difference of 2 groups. P value with less than 0.05 was indicated as significant difference.