Blood specimens (5 ml) were collected from 49 patients with osteosarcoma and 42 healthy volunteers who were admitted to Guangzhou General Hospital of Guangzhou Military Command (Guangzhou, China) between September 2015 and June 2018. Blood was placed in anticoagulation tubes and centrifuged at room temperature for 15 min at 2,000 × g to collect the plasma. The inclusion criteria of patients with osteosarcoma were as follows: i) Osteosarcoma was diagnosed by pathological examinations; ii) osteosarcoma stage I or II; iii) patients who were diagnosed and treated for the first time; and iv) patients who were willing to participate in the study. The exclusion criteria were as follows: i) Presence of co-morbidities; and ii) patients who were treated for other clinical disorders in the three months prior to admission. The patient group included 26 males and 23 females, with an age range of 12–33 years and a mean age of 19.3±4.2 years. The control group was composed of 23 males and 19 females, with an age range of 13–30 years and a mean age of 18.8±4.7 years. The two groups had similar gender and age distributions. Ethical approval was obtained from the Ethics Committee of Guangzhou General Hospital of Guangzhou Military Command. The study followed the tenets of the Declaration of Helsinki, and written informed consent was obtained from all patients and controls after the nature and possible consequences of the study were explained.

The human osteosarcoma cell lines MG-63 and U2OS were used in the current study. The two cell lines were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). Eagle's Minimum Essential Medium (cat no. 30-2003; ATCC) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Sangon Biotech Co., Ltd., Shanghai, China) was used to cultivate cells in an incubator (37°C, 5% CO₂).

Total RNA was extracted from plasma and cells using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and miRNAs were extracted from plasma and cells using the miRNeasy Mini kit (Qiagen GmBH, Hilden, Germany), according to the manufacturer's protocol. An Applied Biosystems™ High-Capacity cDNA Reverse Transcription kit (Applied Biosystems; Thermo Fisher Scientific, Inc.) and a miScript II RT kit (Qiagen GmBH) were used for reverse transcription. qScript One-Step RT-qPCR kit (Quanta Bisciences, Inc., Beverly, MA, USA) and mirVana qRT-PCR miRNA Detection kit (Thermo Fisher Scientific, Inc.) were used to perform qPCR reactions in an ABI 7500 System (Applied Biosystems; Theremo Fisher Scientific Inc.). The miRNA-21 primer pair was purchased from Sigma-Aldrich (cat. no. MIRAP00047; Merck KGaA, Darmstadt, Germany). The following primer pairs and the miRNA-21 primers were used for the qPCR: lncRNA-NEF forward, 5′-CTGCCGTCTTAAACCAACCC-3′ and reverse, 5′-GCCCAAACAGCTCCTCAATT-3′; β-actin forward, 5′-GACCTCTATGCCAACACAGT-3′ and reverse, 5′-AGTACTTGCGCTCAGGAGGA-3′; U6 forward, 5′-CTCGCTTCGGCAGCACA3′ and reverse, 5′-AACGCTTCACGAATTTGCGT-3′. The aforementioned primers were designed and synthesized by Shangai GenePharma Co., Ltd. (Shanghai, China). The following thermocycling conditions were used for the qPCR: Initial denaturation at 95°C for 55 sec, 40 cycles of 95°C for 22 sec and 60°C for 44 sec. mRNA levels were quantified using the 2^−ΔΔCq method (14). miRNA-21 expression was normalized to U6, while lncRNA-NEF was normalized to β-actin.

MISSION^® microRNA Mimic hsa-miR-21* (cat no. HMI0372) and MISSION^® miRNA, Negative Control 1 (cat no. HM0002) were purchased from Sigma-Aldrich; Merck KGaA. lncRNA-NEF expression pIRES vectors and empty pIRES vectors were purchased from Shanghai GenePharma Co., Ltd. lncRNA-NEF sequence refers to a previous study (13). Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) was used to transfect 5×10⁵ cells with miRNA (50 mM) and vectors (15 mM). Transfection was performed according to the manufacturer's protocol. Untransfected cells were used as control (C) cells. Cells transfected with MISSION^® miRNA, Negative Control 1 or empty vectors were used as negative control (NC) cells. Transfection efficacy was determined by RT-qPCR. Cells were harvested 24 h following transfection for subsequent experimentation.

Cell migration and invasion were quantified when lncRNA-NEF and miRNA overexpression rates reached 200–250% following 24 h transfection. Cultrex^® 96-well cell migration/invasion chambers (8 µm) were purchased from Bio-Techne (Minneapolis, MN, USA). The assays were performed according to manufacturer's protocol. Briefly, serum-free Eagle's Minimum Essential Medium was used to prepare single cell suspensions (5×10⁴ cells/ml). A total of 5×10³ cells in 100 µl were transferred to the upper chamber of each well, and the lower chamber was filled with Eagle's Minimum Essential Medium supplemented with 20 % FBS. Cells were maintained in an incubator (37°C, 5% CO₂) for 2 h. Membranes were subsequently stained with 0.5% crystal violet (Sigma-Aldrich; Merck KGaA) at 25°C for 20 min. The invasion assay was performed in the same way as the migration assay; however, the upper chamber was pre-coated with Matrigel (cat no. 356234; Millipore; Merck KGaA) at room temperature for 6 h prior to experimentation.

All experiments were repeated three times and data are presented as the mean ± standard deviation. All statistical analyses were performed using GraphPad Prism 6 software (GraphPad Software Inc., La Jolla, CA, USA). Correlations between plasma lncRNA-NEF and miRNA-21 were analyzed by Pearson's correlation test. Receiver operating characteristic (ROC) curve analysis was performed using patients with osteosarcoma as true positive cases and healthy controls as true negative cases. Comparisons between two groups were performed using the Student's t-test and comparisons among multiple groups were performed by a one-way analysis of variance followed by the Tukey test. P<0.05 was considered to indicate a statistically significant difference.

RT-qPCR results revealed that, compared with the healthy control group, plasma levels of lncRNA-NEF were significantly decreased in patients with osteosarcoma (P<0.05; Fig. 1A). By contrast, plasma levels of miRNA-21 were significantly increased in patients with osteosarcoma compared with healthy controls (P<0.05; Fig. 1B). These data suggest that lncRNA-NEF and miRNA-21 may be implicated in osteosarcoma.

To evaluate the diagnostic value of lncRNA-NEF and miRNA-21 for osteosarcoma, ROC curve analysis was performed with patients with osteosarcoma as true positive cases and healthy controls as true negative cases. For plasma lncRNA-NEF, the area under the curve (AUC) was 0.9176, with a standard error of 0.02793 and 95% confidence interval (CI) of 0.8629–0.9724 (Fig. 2A). For plasma miRNA-21, the AUC was 0.9009, with a standard error of 0.02863 and 95% CI of 0.8457–0.9662 (Fig. 2B).

Correlations between plasma lncRNA-NEF and miRNA-21 in patients with osteosarcoma and healthy controls were analyzed by the Pearson's correlation test. Results revealed that plasma levels of lncRNA-NEF and miRNA-21 were significantly and reversely correlated in patients with osteosarcoma (P<0.05; Fig. 3A). In addition, a significantly reverse correlation between plasma levels of lncRNA-NEF and miRNA-21 was demonstrated in healthy controls (P<0.05; Fig. 3B).

The reverse correlation between plasma lncRNA-NEF and miRNA-21 suggests the possibility of interactions between them. To further explore these potential interactions, lncRNA-NEF expression vectors and miRNA-21 mimics were transfected into the human osteosarcoma cell lines MG-63 and U2OS. lncRNA-NEF and miRNA-21 expression was subsequently detected using RT-qPCR (Fig. 4). Compared with the C and NC groups, lncRNA-NEF overexpression significantly decreased expression of miRNA-21 in the two cell lines (P<0.05; Fig. 4A). By contrast, miRNA-21 overexpression resulted in no significant effects on the expression of lncRNA-NEF (P<0.05; Fig. 4B).

Transwell migration and invasion assays were performed to investigate the effects of lncRNA-NEF and miRNA-21 overexpression on the migration and invasion abilities of MG-63 and U2OS human osteosarcoma cell lines. Compared with the C, control miRNA and empty vector groups, lncRNA-NEF overexpression significantly inhibited and miRNA-21 overexpression significantly promoted migration (P<0.05; Fig. 5A) and invasion (P<0.05; Fig. 5B) of MG-63 and U2OS human osteosarcoma cell lines. Compared with cells with lncRNA-NEF overexpression alone, cells with lncRNA-NEF and miRNA-21 overexpression exhibited significantly reduced migration (P<0.05; Fig. 5A) and invasion (P<0.05; Fig. 5B) abilities.