OS tumor tissue and matched adjacent normal tissue were collected from 22 patients diagnosed with primary OS at the First Affiliated Hospital of the Medical College, Shihezi University. This study was performed with the approval of the Research Medical Ethics Committee of the First Affiliated Hospital of the Medical College, Shihezi University. Each patient signed written informed consent prior to enrolling in this medical study.

Human OS cell lines (U2OS, MG-63, Saos-2, and 143B) and the human normal osteoblastic cell line FOB1.19, together with Human Embryonic Kidney 293 cells (HEK293), were obtained from American Type Culture Collection (ATCC, Rockville, MD, USA). U2OS and 143B cells were cultured in RPMI-1640 (Gibco Co., New York, NY, USA) medium supplemented with 10% fetal bovine serum (FBS, Invitrogen, Carlsbad, CA, USA). MG-63 were grown in MEM medium (Gibco) containing 10% FBS (Invitrogen). Saos-2 cells were cultured in McCoy's 5A medium (Sigma-Aldrich, St. Louis, MO, USA) containing 15% FBS (Invitrogen). The human normal osteoblastic cell line hFOB 1.19 was maintained in DMEM/F-12 medium (Gibco) supplemented with 10% FBS (Invitrogen). HEK293 cells were maintained in DMEM (Gibco) medium containing 10% FBS (Invitrogen). All cells were maintained in humidified incubator containing 5% CO₂ at 37℃.

Fresh OS tumor tissue obtained from patients with primary spontaneous OS was washed using sterile phosphate-buffered saline three times and then minced into small tumor pieces. Then, tumor samples were digested in digestion medium [DMEM/F-12 medium (Gibco) containing 0.1 mg/mL of hyaluronidase (Sigma-Aldrich), 2 mg/mL of collagenase A (Sigma-Aldrich), 60 U/mL of nystatin (Sigma-Aldrich), and 0.1 mg/mL of gentamicin (Sigma-Aldrich)] for almost 50 min at 37℃. Then, cells were cultured in DMEM/F12 medium (Gibco) supplemented with 10% FBS (Invitrogen) in a humidified incubator containing 5% CO₂ at 37℃.

Small interference RNA (siRNA) targeting TUG1 (si-TUG1) and its scramble control (si-NC), miR-132-3p mimic and its negative control (miR-NC), miR-132-3p inhibitor (anti-miR-132-3p) and its control anti-miR-NC, and SOX4 siRNA (si-SOX4) and its scramble control (si-NC) were obtained from GenePharma Co., Ltd. (Shanghai, China). TUG1 and SOX4 cDNA sequences were cloned into pcDNA3.1 (Invitrogen) to produce TUG1 and SOX4 overexpression vectors (pcDNA3.1-TUG1 and pcDNA3.1-SOX4), named TUG1 and SOX4.

The MG-63, U2OS, and primary OS cells were transfected with miRNA, siRNA, miRNA inhibitors, and plasmids by Lipofe-ctamine 2000 (Invitrogen) according to the manufacturer's instructions, and the cells were harvested at the indicated time points after transfection.

Total RNA from the tissues and cells was obtained by TRIzol solution (Invitrogen) in accordance with the manufacturer's protocols. cDNA was synthesized by the PrimeScript RT reagent kit (TaKaRa, Tokyo, Japan). Quantitative PCR analysis of TUG1 was performed using the SYBR® Premix Ex Taq™ reagent (TaKaRa), with β-actin as an endogenous control. The reverse transcription and quantification of miR-132-3p was carried out using the miDETECTA Track™ miRNA qRT-PCR Starter kit (RiboBio, Guangzhou, China), referring to the manufacturer's protocols, with U6 snRNA as an internal normalization reference.

The real-time quantitative PCR (RT-qPCR) primers sequences were as follows: TUG1, 5′-CTGAAGAAAGGCAACATC-3′ (forward) and 5′-GTAGGCTACTACAGGATTTG-3′ (reverse); β-actin, 5′-AGTGTGACGTGGACATCCGCAAAG-3′ (forward) and 5′-ATCCACATCTGCTGGAAGGTGGAC-3′ (reverse). Reverse transcription primers, 5′-GTCGTATCCAGTGCAG GGTCCGAGGTATTCGCACTGGATACGACCGACCATG-3′ (miR-132-3p); 5′-AAAATATGGAACGCTTCACGAATTTG-3′ (U6). Quantification primers, miR-132-3p, 5′-GCGCGCGTAACAGTCTACAGC-3′ (forward) and 5′-GTCGTATCCAGTGCAGGGTCC-3′ (reverse); U6, 5′-CTCGCTTCGGCAGCACATATACT-3′ (forward) and 5′-CGCTTCACGAATTTGCGTGT-3′ (reverse).

Cells from different treatment conditions were obtained 48 hours after transfection, and western blot assays were carried out with the SOX4 and β-actin antibodies (Santa Cruz Biotechnology, Dallas, TX, USA) according to manufacturer's protocols. Briefly, MG-63 and U2OS cells were lysed using RIPA lysis buffer (Beyotime, Shanghai, China) containing cocktail (Roche, Mannheim, Germany) to obtain the whole protein. The protein concentration was measured using Pierce™ BCA Protein Assay Kit (Thermo scientific, Rockford, IL, USA). Then, equal weights (50 µg) of proteins were separated using SDS-PAGE gel, followed by being transferred to PVDF membranes (Millipore, Billerica, MA, USA). Next, the membranes were blocked for 1 h with 5% skimmed milk and incubated with appropriate concentrations of antibodies against SOX4 and β-actin antibodies (Santa Cruz Biotechnology) overnight at 4℃. On the next day, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody for another 1 h. Finally, protein signals were detected using the BeyoECL Plus kit (Beyotime) and quantified with Image J software (National Institutes of Health, Bethesda, MD, USA).

MG-63, U2OS, and primary OS cells were seeded into 96-well plates and transfected using Lipofectamine 2000 reagent. Then, cell proliferation was assessed using the XTT Cell Proliferation Assay Kit (Abnova, Taipei, Taiwan) following the manufacturer's protocols. Briefly, 10 µL of XTT was added into 96-well plates at indicated time points (0, 24, 48, and 72 h) after transfection and then incubated for another 3 h at 37℃ incubator. Finally, absorbance was measured at the wavelength of 450 nm.

Partial DNA sequences of TUG1 and SOX4 3'-UTR containing wild-type (WT) or mutant (MUT) miR-132-3p binding sites were amplified by PCR and then cloned into pmiR-RB-REPORT™ vectors (RiboBio) to produce WT-TUG1, MUT-TUG1, WT-SOX4-3'UTR, and MUT-SOX4-3'UTR reporter plasmids. Then, constructed reporter plasmids were respectively co-transfected with miR-132-3p or miR-NC into 293T cells using Lipofectamine 2000 (Invitrogen). Then, the luciferase activity in cells from different treatment conditions was measured by the Dual-Luciferase® Reporter Assay kit (Promega, Madison, WI, USA) according to manufacturer's instructions.

After transfection for 48 h, transfected cells were treated using the AnnexinV-FITC/PI Apoptosis Detection Kit (BD Biosciences, Franklin Lakes, NJ, USA), referring to manufacturer's instructions, and then, flow cytometry (FACScan; BD Biosciences) was used to determine cell apoptosis rates.

MG-63 and U2OS cells were collected 48 h after transfection, and caspase-3 activity in these cells were measured using the Caspase 3 Activity Assay Kit (Beyotime), referring to the manufacturer's protocols.

All data were obtained from at least three independent experiments and are reported as means±standard deviations. One-way analysis of variance or Student's t-test was conducted to analyze difference therein. Differences were regarded as statistically significant when p<0.05.

To determine the expression pattern of TUG1 in OS tissues, 22 pairs of OS tumor tissues and correspondingly adjacent normal tissues were collected, followed by the detection of TUG1 levels using RT-qPCR assays. TUG1 expression was remarkably upregulated in 17 (77.3%) OS tumor tissues, compared to correspondingly normal tissues, together with a slight downregulation in the remaining (22.7%) OS tumor tissues (Fig. 1A). The overall expression of TUG1 was upregulated in OS tumor tissues, compared with normal tissues (Fig. 1B). RT-qPCR results revealed that TUG1 was highly expressed in OS cell lines (MG-63, U2OS, Saos-2, and 143B), compared with the human osteoblast cell line hFOB 1.19 (Fig. 1C). All these results indicated that the expression of TUG1 in OS tumors and cells is significantly upregulated, which is in accordance with previous results.1415

To investigate the roles of TUG1 in OS cell lines, TUG1 overexpression plasmid (pcDNA3.1-TUG1, TUG1) and siRNA of TUG1 (si-TUG1) were constructed or synthesized. Then, the transfection efficiency of TUG1 and si-TUG1 was respectively assessed by RT-qPCR assays. As shown in Fig. 1D, the transfection of pcDNA3.1-TUG1 resulted in a noticeable upregulation of TUG1 expression, compared with the negative control (vector), in MG-63 cells. We also observed that TUG1 expression was strikingly downregulated in U2OS cells transfected with si-TUG1 in comparison with the transfection of non-specific siRNA (si-NC) (Fig. 1E). These results revealed that the TUG1 overexpression plasmid and TUG1 siRNA could exert its function in the following experiments.

Next, the effects of TUG1 on the proliferation and apoptosis of MG-63 and U2OS cells were further investigated. TUG1 and si-TUG1 were respectively transfected into MG-63 and U2OS cells, and cells proliferation was assessed at 0, 24, 48, and 72 h by XTT assays (Fig. 1F and G). The results revealed that TUG1 overexpression significantly promoted MG-63 cell proliferation (Fig. 1F) and that TUG1 knockdown markedly suppressed U2OS cell proliferation (Fig. 1G). The effect of TUG1 on U2OS cell apoptosis was confirmed by the flow cytometry and caspase-3 activity detection. As expected, the depletion of TUG1 by si-TUG1 induced increases in apoptosis (Fig. 1H) and caspase-3 activity (Fig. 1I) in U2OS cells.

Emerging evidence indicates that lncRNAs act as ceRNAs of miRNAs to regulate target mRNAs expression.89 Therefore, bioinformatics prediction analysis was performed by the miRcode online website to search for potential target miRNAs of TUG1. Among candidate miRNAs, miR-132-3p was chosen due to its anti-tumor effect in OS (Fig. 2A).1617 For validation thereof, WT-TUG1 (containing the putative miR-132-3p binding sites) and MUT-TUG1 (putative miR-132-3p binding sites mutated to the indicated sequences) luciferase reporter plasmids were constructed. Then, the effect of miR-132-3p on the luciferase activity of the WT-TUG1 or MUT-TUG1 reporter plasmid was detected, and the results indicated that the introduction of miR-132-3p mimic significantly inhibits the luciferase activity of WT-TUG1 reporter, compared with miR-NC, whereas miR-132-3p had no effect on the luciferase activity of MUT-TUG1 reporter in HEK293 cells (Fig. 2B). These finding suggested that the putative binding sites were indeed essential for the direct interaction of miR-132-3p and TUG1. RT-qPCR assays further demonstrated that enforced expression of TUG1 markedly suppressed miR-132-3p expression in MG-63 cells, while TUG1 knockdown facilitated miR-132-3p expression in U2OS cells (Fig. 2C). Thus, TUG1 inhibits the expression of miR-132-3p by direct interaction in MG-63 and U2OS cells.

Next, we further explored whether miR-132-3p could influence the effect of TUG1 on proliferation and apoptosis in OS cells. XTT assay results indicated that the restoration of miR-132-3p partly abolished the promotion effect of TUG1 overexpression on proliferation in MG-63 cells (Fig. 2D). Also, the introduction of miR-132-3p inhibitor partially abrogated si-TUG1-mediated anti-proliferative effects in U2OS cells (Fig. 2E). Moreover, the transfection of miR-132-3p inhibitor resulted in a marked decrease in apoptosis rate (Fig. 2F) and caspase-3 activity (Fig. 2G) in U2OS cells upon transfection of si-TUG1, compared to U2OS cells transfected with si-TUG1 and control (anti-miR-NC). Collectively, si-TUG1 exerted its anti-proliferation and pro-apoptosis effects partly by targeting miR-132-3p in OS cells.

As is known, miRNAs can exert regulatory roles by affecting target gene expression. Hence, TargetScan online was employed to search for genes that had a chance to interact with miR-132-3p. Among candidate genes, SOX4 has been identified as an oncogene in OS (Fig. 3A),161819 and was chosen for further study. To validate whether the putative binding sites of SOX4-3'UTR and miR-132-3p are important for the interaction between miR-132-3p and SOX4, dual luciferase reporter assays and mutation assays were performed. Then, constructed WT-SOX4-3'UTR or MUT-SOX4-3'UTR reporter was co-transfected with miR-132-3p or miR-NC into HEK293 cells, and their luciferase activities were measured 48 h after transfection. As shown in Fig. 3B, the introduction of miR-132-3p mimic brought about a notable reduction in luciferase activity of the WT-SOX4-3'UTR reporter, compared with negative control (miR-NC). However, miR-132-3p had no effect on the luciferase activity of MUT-SOX4-3'UTR reporter (Fig. 3B). These findings indicated that miR-132-3p interacts with SOX4-3'UTR via the putative binding sites. Next, western blot assays further suggested that the ectopic expression of miR-132-3p decreases SOX4 protein levels, compared with the negative control miR-NC, in MG-63 cells (Fig. 3C). Conversely, miR-132-3p deficiency resulted in a marked increase in SOX4 protein expression, compared with control (anti-miR-NC), in U2OS cells (Fig. 3C). Overall, these results suggest that SOX4 is a target of miR-132-3p in OS cells.

Then, the effects of miR-132-3p and SOX4 on proliferation and apoptosis were further investigated in MG-63 and U2OS cells. XTT assays showed that the introduction of miR-132-3p mimic remarkably suppressed MG-63 cell proliferation, compared with negative control (miR-NC) (Fig. 4A). As expected, the transfection of miR-132-3p inhibitor increased U2OS cell proliferation, compared with negative control (anti-miR-NC) (Fig. 4B). Moreover, the overexpression of miR-132-3p gave rise to a notable increase in apoptosis rate (Fig. 4C) and caspase-3 activity (Fig. 4D) in MG-63 cells. In restoration assays, the reintroduction of SOX4 partly reversed miR-132-3p-mediated anti-proliferation (Fig. 4A) and pro-apoptosis (Fig. 4C and D) effects in MG-63 cells. Also, the introduction of si-SOX4 weakened the promotive effect of miR-132-3p inhibitor on proliferation in U2OS cells (Fig. 4B). Collectively, miR-132-3p was deemed to inhibit proliferation and induce apoptosis by targeting SOX4 in OS cells.

Interestingly, TUG1 and SOX4 shared the same binding sites in the process of interacting with miR-132-3p, indicating that TUG1 might act as a ceRNA to regulate the expression of miR-132-3p and SOX4. Western blot assays demonstrated that TUG1 overexpression markedly improved SOX4 protein levels, while this effect of TUG1 on SOX4 expression was dramatically abated by miR-132-3p in MG-63 cells (Fig. 5A). On the contrary, TUG1 knockdown resulted in a significant reduction in SOX4 protein expression and miR-132-3p inhibitor reversed the inhibition effect of si-TUG1 on SOX4 expression in U2OS cells (Fig. 5B). Taken together, TUG1 might serve as a ceRNA of miR-132-3p to increase SOX4 expression in OS cells.

Primary OS cells were isolated from OS patients to further explore whether TUG1 exerts its carcinogenic effect by regulating miR-132-3p/SOX4 in human OS patients. First, we demonstrated that TUG1 is highly expressed in human primary OS cells, compared with the human normal osteoblastic cell line FOB1.19 (Fig. 6A). Next, the transfection efficiency of pcDNA-TUG1 and si-TUG1 was detected in human primary OS cells. As shown in Fig. 6B, the transfection of pcDNA-TUG1 overexpression plasmid strikingly promoted TUG1 expression and the introduction of si-TUG1 markedly inhibited TUG1 expression in human primary OS cells. Then, the effects of TUG1 overexpression and silencing on cell proliferation were further examined in human primary OS cells. The results showed that cell proliferation capacity was elevated in TUG1-overexpressed primary OS cells, which was markedly reduced in TUG1-depleted primary OS cells (Fig. 6C and D). Moreover, we further demonstrated that TUG1 knockdown facilitated primary OS cell apoptosis, reflected as increases in apoptosis rate and caspase-3 activity in si-TUG1-transfected cells (Fig. 6E and F). As demonstrated above, TUG1 exerted its carcinogenic effect by downregulating miR-132-3p expression and upregulating SOX4 expression in OS cell lines. Thus, the effects of TUG1 on miR-132-3p and SOX4 expression were explored in primary OS cells. The results revealed that enforced expression of TUG1 suppresses miR-132-3p expression and facilitates SOX4 expression in primary OS cells (Fig. 6G and H). Conversely, TUG1 silencing induced a notable increase in miR-132-3p level and a marked reduction in SOX4 expression in primary OS cells (Fig. 6G and H). Overall, these data indicated that the knockdown of TUG1 suppresses proliferation and promotes apoptosis by regulating the miR-132-3p/SOX4 axis in primary OS cells.