TPX2 and microRNA (miRNA/miR)-29c-3p expression levels were explored using the Gene Expression Profiling Interactive Analysis (GEPIA) (http://gepia.cancer-pku.cn/index.html) and OncoLnc (http://www.oncolnc.org/) databases, respectively. In GEPIA, the term ‘TPX2’ was used in the ‘Gene’ field and ‘SARC’ was used in the ‘Datasets Selection’ field. Differential expression of TPX2 was defined with the following significance cutoff levels: |Log2FC|>1 and P<0.01. In OncoLnc, ‘SARC’ was selected in the ‘miRNA’ column, and the data was downloaded. The information for miR-29c-3p was obtained. The Kaplan Meier plotter (http://kmplot.com/analysis/) was used to analyze the overall survival of patients with different levels of TPX2 and miR-29c-3p expression in sarcoma. TargetScan (http://www.targetscan.org/vert_71/) was used to explore the potential association between TPX2 and miR-29c-3p.

Between January 2008 and December 2018 (age range, 2–54 years; average age, 19.47±3.30 years), a total of 52 pairs of primary osteosarcoma and adjacent noncancerous tissues were obtained from patients who had undergone a wide resection of osteosarcoma at the First People's Hospital of Lianyungang (Lianyungang, China). Inclusion criteria were as follows: Osteosarcoma patients without radiotherapy or chemotherapy before surgery. Exclusion criteria were as follows: i) Osteosarcoma patients who received radiotherapy or chemotherapy before surgery; and ii) patients who refused to sign the informed consent form. Morphologically normal tissues that were <5 cm from the cancerous tissues were used as adjacent noncancerous tissues. All samples were frozen at −80°C until further use. The histological diagnosis of osteosarcoma conformed to the World Health Organization's histological criteria for osteosarcoma (16), as confirmed by two professional pathologists.

The Ethics Committee of the First People's Hospital of Lianyungang approved all protocols, which complied with the Declaration of Helsinki (December 2018; approval no. 20180203). Written informed consent was obtained from all patients and/or their legal guardians.

Human osteosarcoma MG63 and U2OS cell lines, and the human normal osteoblastic hFOB cell line were purchased from The Cell Bank of Type Culture Collection of the Chinese Academy of Sciences. All cells were cultured in DMEM (Invitrogen; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 100 µg/ml streptomycin at 37°C in a humidified incubator containing 5% CO₂.

RNA isolation and RT-qPCR were performed as described previously (17). TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) was used to extract RNA from the three cell lines and the tissues. Next, the EasyScript One Step gDNA Removal and cDNA Synthesis SuperMix (Beijing TransGen Biotech Co., Ltd.) was used to acquire the first strand cDNA according to the manufacturer's instructions. Next, qPCR was performed using SYBR-Green Master Mix kit (Roche Diagnostics GmbH) with an Applied Biosystems 7900 Real-Time PCR System (Thermo Fisher Scientific, Inc.). The reaction conditions were as follows: Pre-denaturation at 95°C for 15 min, followed by 35 cycles of denaturation at 95°C for 15 sec, annealing at 58°C for 30 sec and extension at 72°C for 5 min, and then a final extension at 72°C for 15 min. Expression levels of RNA were calculated based on the comparative 2^−ΔΔCq method (18). U6 was used as an endogenous control for miR-29c-3p, and β-actin was used as an endogenous control for TPX2. All experiments were performed in triplicate. The primers used are listed in Table I.

Knockdown experiments for TPX2 were performed using siRNA oligonucleotides purchased from Shanghai GenePharma Co., Ltd. The TPX2 siRNA sequences were as follows: Forward, 5′-CCAUUAACCUGCCAGAGAAT-3′ and reverse, 5′-UUCUCUGGCAGGUUAAUGGT-3′. The negative control for siRNA silencing was a non-targeting (scramble) siRNA sequence, with the sequence 5′-UUCUCCGAACGUGUCACGUTT-3′. MG63 and U2OS cells were plated in 6-well plates at a density of 2×10⁵ cells/well and cultured in growth medium until cell confluence reached 70%. Transfection of siRNA (20 nM) was then performed using Lipofectamine^® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Transfection was performed at 37°C for 6 h before replacing the transfection medium with full culture medium. At 48 h post-transfection, cells were harvested for RT-qPCR or western blot analysis.

The MG63 and U2OS cells were seeded in 96-well plates at a density of 1,000 cells per well with 5 replicate wells at 48 h post-siRNA transfection. Cell proliferation was measured using an MTT assay at 0, 24, 48 and 72 h after seeding. Next, 150 µl dimethylsulfoxide was added per well to dissolve the purple formazan. The absorbance was measured at a wavelength of 490 nm using a microplate reader. All proliferation experiments were performed in triplicate.

MG63 and U2OS cells were seeded into 24-well plates. After 24 h of incubation, 6 ng pmirGLO report vector (Shanghai GenePharma Co., Ltd.) carrying the wild-type (wt) or mutant (mut) 3′-untranslated region (3′-UTR) of TPX2 was cotransfected with miR-29c-3p mimics (100 nmol/l) or miR-29c-3p inhibitors (100 nmol/l) into the osteosarcoma cells using a Lipofectamine 2000 kit (Invitrogen; Thermo Fisher Scientific, Inc.). The negative controls for the miRNA mimics and inhibitors were non-targeting sequences. The sequences of the mimics, inhibitors and negative controls were as follows: miR-29c-3p mimic sense, 5′-UAGCACCAUUUGAAAUCGGUUA-3′ and antisense, 5′-UAACCGAUUUCAAAUGGUGCUA-3′; negative control mimic sense, 5′-UCACAACCUCCUAGAAAGAGUAGA-3′ and anti-sense, 5′-UCUACUCUUUCUAGGAGGUUGUGA-3′; miR-29c-3p inhibitor, 5′-UCUACUCUUUCUAGGAGGUUGUGA-3′; and negative control inhibitor, 5′-UAACCGAUUUCAAAUGGUGCUA-3′ (Shanghai GenePharma Co., Ltd.). The amplified TPX2 wt or mut 3′UTR containing the miR-29c-3p target sites was inserted into the pMIR-GLO luciferase reporter vectors (Shanghai GenePharma Co., Ltd.). At 48 h post-transfection, luciferase activities were examined with a dual-luciferase reporter system (Shanghai GenePharma Co., Ltd.) and normalized with Renilla luciferase activity using SpectraMax i3 (Molecular Devices LLC).

Western blotting analysis was performed as previously reported (17). MG63 and U20S cell lines were analyzed. Primary antibodies involved in this assay were as follows: Anti-TPX2 (1:1,000; catalog no. ab71816), anti-HSP90 (1:5,000; catalog no. ab203085), anti-AKT (1:500; catalog no. ab8805), anti-p-AKT (1:1,000; catalog no. ab18206), anti-BRCA1 (1:1,000; catalog no. ab245330) and anti-GAPDH (1:1,000; catalog no. ab9485) (all Abcam). The secondary antibody was anti-rabbit IgG (1:4,000; catalog no. ab150077; Abcam). Primary antibodies were incubated at 4°C overnight, and secondary antibody was incubated for 1 h at room temperature. The protein bands were detected by a chemiluminescence detection system (Beyotime Institute of Biotechnology). Pierce ECL Western Blotting Substrate (cat. no. 32109; Thermo Fisher Scientific, Inc.) was used to visualize the bound antibodies. Image analysis was performed using ImageJ software (version 1.42; National Institutes of Health).

Statistical analyses were performed using GraphPad Prism 8.0 (GraphPad Software, Inc.). Values are presented as the mean ± standard deviation. The significance of differences between two groups was assessed by paired or unpaired Student's t-test. Pearson's χ² and Fisher's exact tests were performed to assess the categorical variables. Prognostic significance was determined by Kaplan-Meier and log-rank analyses. A receiver operating characteristic (ROC) curve was calculated to determine the area under the curve (AUC) for biomarkers. Pearson's correlation was used to evaluate the strength of linear correlation between two continuous variables. P<0.05 was considered to indicate a statistically significant difference.

By analysis of the GEPIA public database, the relative expression of TPX2 was found to be significantly higher in sarcoma tissues (n=262) compared with that in normal tissues (n=2) at the mRNA level (Fig. 1A). The OncoLnc database showed that miR-29c-3p was expressed at lower levels in tumor tissues compared with that in normal tissues (Fig. 1B). Kaplan-Meier plotter indicated that patients with high TPX2 expression experienced poor overall survival (Fig. 1C), while those with high miR-29c-3p expression experienced favorable overall survival (Fig. 1D). In 52 pairs of osteosarcoma tissues and adjacent normal tissues, the expression of TPX2 was significantly upregulated in tumor tissues compared with that in paired adjacent normal tissues (Fig. 2A). Additionally, miR-29c-3p expression was downregulated in osteosarcoma (Fig. 2B). Furthermore, the TPX2 expression level was measured in MG63 and U2OS cell lines. The RT-qPCR analysis showed significantly higher mRNA levels of TPX2 in these tumor cells compared with that in normal hFOB cells (Fig. 2C). By contrast, miR-29c-3p expression was downregulated in the osteosarcoma cell lines (Fig. 2D). Pearson's correlation analysis disclosed a significant negative correlation between TPX2 and miR-29c-3p expression in osteosarcoma (P=0.002, r=−0.42) (Fig. 2E).

The optimal cut-off value of TPX2 was determined by ROC analysis based on its expression level (P<0.001, AUC=0.83) (Fig. 2F). The 52 patients were consequently divided into two groups: TPX2 high expression (n=28) and TPX2 low expression (n=24). The associations between TPX2 expression and various clinicopathological characteristics are shown in Table II. The results indicated that high TPX2 expression level was significantly associated with tumor size (P=0.019), clinical stage (P=0.002) and distant metastasis (P=0.018), compared with low expression level. However, no association was found with other clinical parameters, such as age, sex and anatomical location. Kaplan-Meier and log-rank test analysis revealed that high TPX2 expression was significantly associated with a poor prognosis (P=0.018) (Fig. 2G).

TPX2 siRNA plasmids were transfected into MG63 and U2OS osteosarcoma cell lines to knockdown TPX2 expression. RT-qPCR was then used to explore the efficiency of the knockdown (Fig. 3A and B). The transfection efficiency was also confirmed by western blotting (Fig. 3C). MTT assays results indicated that TPX2 knockdown inhibited osteosarcoma MG63 and U2OS cell line proliferation (Fig. 3D and E).

In osteosarcoma MG63 and U2OS cell lines, after transfection of miR-29c-3p mimics or inhibitors, the efficiency was confirmed by RT-qPCR (Fig. 4A-D). MTT assays results indicated that miR-29c-3p mimics could significantly decrease the proliferation of the MG63 and U2OS cell lines, while miR-29c-3p inhibitors could significantly promote MG63 and U2OS cell line proliferation (Fig. 4E-H).

Using the TargetScan database, it was determined that the TPX2 3′UTR included a putative miR-29c-3p binding site (Fig. 5A). This notion was subsequently validated using a luciferase reporter assay. MG63 and U2OS cell lines were cotransfected with luciferase reporter vectors containing TPX2-3′UTR-wt or TPX2-3′UTR-mut, along with miR-29c-3p mimics or inhibitors. Luciferase reporter gene assay revealed that miR-29c-3p mimics significantly inhibited luciferase activity in the TPX2-wt group (Fig. 5B and C) and that miR-29c-3p inhibitors promoted luciferase activity in the TPX2-wt group (Fig. 5D and E). Additionally, TPX2 expression decreased in miR-29a-5p mimics, while it increased in the miR-29c-3p inhibitors group compared with the NC (Fig. 5F and G). Taken together, these data show that TPX2 is a target gene of miR-29c-3p and that a negative correlation exists between miR-29c-3p and TPX2 in osteosarcoma.

The findings showed that, compared with that in the NC group, TPX2 expression in MG63 and U2OS cells transfected with miR-29c-3p inhibitors increased; however, when cotransfected with TPX2 siRNA and miR-29c-3p inhibitors, the expression of TPX2 was significantly decreased compared with that in the group without siRNA (Fig. 5H and I). MTT assays results indicated that the knockdown of TPX2 significantly reversed miR-29c-3p inhibitor-mediated promotion of cell proliferation in the osteosarcoma cell lines (Fig. 5J and K).

The AKT signaling pathway is an important signaling pathway that controls the proliferation of cancer cells (Fig. 6A). A positive correlation was found between TPX2 and the three key genes in the AKT signaling pathway using the GEPIA database (Fig. 6B-D). Western blotting was used to detect the protein expression levels of HSP90, AKT and BRCA1 in osteosarcoma cells after TPX2 silencing, in order to determine the effect of TPX2 on the AKT pathway. It was found that the expression of the aforementioned key genes was decreased when TPX2 was knocked down in U2OS and MG63 cells (Fig. 6E). Taken together, the results showed that TPX2 regulated by miR-29c-3p induces cell proliferation in osteosarcoma via the AKT signaling pathway (Fig. 6F).