MG63, HOS, SaO2 and U2-OS osteosarcoma cell lines were purchased from the American Type Culture Collection (ATCC, Vanassas, MA, USA). HMGN2 lentivirus vector, negative control vector GFP and virion-packaging elements were purchased from Genechem (Shanghai, China). All the antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

TRIzol reagent, Lipofectamine 2000, Double stain apoptosis detection kit (Annexin V-FITC/PI), Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco Invitrogen Corporation (Grand Island, NY, USA). 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfo-phenyl)-2H-tetrazolium (WST-8) was purchased from Beyotime (Beyotime, Haimen, China) and SYBR-Green master mixture was purchased from Takara (Otsu, Japan).

Human-HMGN2 cDNA was prepared by RT-PCR (Fig. 1A) and inserted into the BamHI and XhoI restriction sites of the pGC-FU-3FLAG vector plasmid (Addgene, Cambridge, MA, USA). For the stable overexpression of HMGN2, HEK293T cells were plated in 75-cm² culture flasks and transfected with 10 μg HMGN2 or GFP (control) lentivirus vectors. The medium was changed and the viral supernatant was harvested 48 h later. Viral-containing medium was collected and passed through 0.45-μm syringe filters. SaO2 and U2-OS cells were incubated with the lentivirus supernatant for 24 h and selected with 2 μg/ml puromycin (Sigma, St. Louis, MO, USA) according to the manufacturer’s instructions. The clone in which the HMGN2 lentivirus vectors transfected was designated as the HMGN2 group, the negative control GFP transfected vectors was designated as the GFP group and SaO2 and U2-OS osteosarcoma cells were designated as the CON group.

To quantitatively determine the expression level of HMGN2 in osteosarcoma cell lines, HMGN2 mRNA expression was determined by RT-qPCR using SYBR-Premix Ex Taq (Takara, Japan) and an ABI Prism 7500 sequence detection system (Applied Biosystems, USA). Total RNA of each clone was extracted with TRIzol according to the manufacturer’s instructions. The genes were amplified using specific oligonucleotide primers and human β-actin gene was used as an endogenous control. The PCR primer sequences used were: HMGN2, forward: 5′-CCAGCCATCAGCCATGAGGGT-3′ and reverse: 5′-GGAGCCCTTTCTGAATCCGCA-3′); β-actin, forward: 5′-GCGGGAAATCGTGCGTGACATT-3′ and reverse: 5′-GGCAGATGGTCGTTTGGCTGAATA-3′. Data were analyzed using the comparative Ct method (2^−ΔΔCt). Three separate experiments were performed for each clone.

Treated cells were collected and extracted using an assay kit (Beyotime, China) and the concentration was determined by using a Bio-Rad protein assay (Hercules, CA, USA). Equal amounts of cell extracts were separated on SDS-PAGE gels according to the molecular weight of the tested proteins. Separated protein bands were transferred onto polyvinylidene fluoride membranes and blocked in 5% skimmed milk powder. The primary antibodies against HMGN2, cyclin E, cyclin D1, PCNA, and Ki-67 (all from Santa Cruz Biotechnology) were diluted according to the instructions and incubated overnight at 4°C. Horseradish peroxidase-linked secondary antibodies were added at a dilution ratio of 1:1,000, and incubated at room temperature for 2 h. The membranes were washed with phosphate-buffered saline three times and the immunoreactive bands were visualized using an ECL-Plus kit according to the manufacturer’s instructions [Multi Sciences (Lianke) Biotech Co., Ltd., Hangzhou, China]. The relative protein level in different cell lines was normalized to β-actin concentration. Three separate experiments were performed for each clone.

Cell proliferation was analyzed with the cell count assay using WST-8 kits. Briefly, cells infected with HMGN2 were incubated in 96-well plates at a density of 1×10⁵ cells/well with DMEM with 10% FBS. The cells were treated with 10 μl WST at 6, 12 and 24 h. The color reaction was measured at 570 nm with enzyme immunoassay analyzer (Bio-Rad Laboratories, Hercules, CA, USA). The proliferative activities were calculated for each clone.

For the cell cycle analysis, harvested cells were centrifuged at 1,000 × g for 5 min, washed with phosphate buffered saline and fixed in 70% ethanol overnight. The cells were then treated with 100 μg/ml propidium iodide plus RNase (10 μg/ml) for 30 min. The cell-cycle phase distribution was determined by analytical DNA flow cytometry (FACSCalibur; BD Biosciences, Becton-Dickinson, San Jose, CA, USA) as described by Evans et al (13).

SaO2 and U2-OS cells were plated in each well of a 6-well culture plate and allowed to grow to 90% confluence. Treatment with HMGN2 lentivirus was then performed. The following day, a wound was created using a 10 μl micropipette tip. The migration of cells towards the wound was monitored daily, and images were captured at time intervals of 24 h.

Three mice were injected subcutaneously with 1×10⁸ SaO2 and U2-OS cells in 50 ml of PBS pre-mixed with an equal volume of Matrigel matrix (Becton Dickinson). When the tumor size reached ~5 mm in length, sections were surgically removed, cut to 1-mm³, and re-seeded individually into other mice. When the tumor size reached ~5 mm in length, the mice were randomly assigned to the GFP and HMGN2 groups, in which 15 ml of lentivirus was injected into subcutaneous tumors using a multi-site injection format. The injections were repeated on the third day after initial treatment. The tumor volume was measured every three days with a caliper, using the formula volume = (length × width)²/2.

The results obtained were expressed as the mean ± standard error values from at least three independent experiments. One-way analysis of variance (ANOVA) was used to analyze the differences between groups. The LSD method of multiple comparisons was used when the probability for ANOVA was statistically significant. P<0.05 was considered to indicate statistical significance.

The endogenous expression of HMGN2 in human osteosarcoma SaO2, MG-63, U2-OS and HOS cell lines was evaluated using RT-qPCR and western blot analysis. As shown in Fig. 1A and B, there were different levels of mRNA and protein expression of HMGN2 in SaO2, MG-63, U-2 OS, and HOS cell lines, while the expression levels of HMGN2 were significantly reduced in the SaO2 and U-2 OS cell lines than those in the MG-63 and HOS cell lines.

Since HMGN2 exhibited a low expression in SaO2 and U-2 OS cell lines, the cell lines were selected as the infective objects of HMGN2 lentivirus. The infection efficiencies of HMGN2 (at a multiplicity of infection =50) in SaO2 and U-2 OS cell lines were >80% under fluorescence microscopy (Fig. 2C). To confirm that HMGN2 successfully transfected into SaO2 and U-2 OS cells, western blot analysis was applied. After 48 h following HMGN2 or GFP lentivirus infection and the expression of HMGN2 protein was significantly increased in the HMGN2 group (Fig. 1D).

Deregulated cell proliferation is a hallmark of cancer (16). To determine the effect of HMGN2 overexpression on SaO2 and U-2 OS cell growth, we investigated the proliferative activities by WST assay. Overexpression of HMGN2 significantly reduced the proliferative activities of SaO2 and U-2 OS cells compared with the GFP and CON groups (Fig. 2A). In addition, PCNA and Ki-67, which were indicators for cell proliferation, were examined by western blot assay to determine whether HMGN2 overexpression suppressed cell growth through translational repression. The expression of PCNA protein was significantly decreased in Lenti-HMGN2 group compared with the CON and GFP groups (P<0.01) while Ki-67 did not alter among these groups (Fig. 2B). These data suggested that the overexpression of HMGN2 may inhibit osteosarcoma cell proliferation through the downregulation of PCNA expression.

The cycle distribution of SaO2 and U-2 OS cells was also analyzed. As shown in Fig. 3A, the percentage of untransfected SaO2 and U-2 OS cells in S phase was 52.1 and 58%, respectively, whereas the percentage of SaO2 and U-2 OS cells transfected with HMGN2 in S phase was 35.5 and 33%, respectively. In addition, 10 and 15% of untreated SaO2 and U-2 OS cells were in the G2/M phase compared with 5 and 8% of cells transfected with HMGN2 lentivirus. These results indicated that cell cycle was arrested in G0/G1 phase in HMGN2 group compared with the CON and GFP groups. Subsequently, cyclin D1 and cyclin E, two regulators of cell cycle progression from G1 to S phase, were assessed by western blotting (17). The results showed that only HMGN2 led to a decreased level of cyclin D1, whereas no significant changes were identified in the levels of cyclin E (Fig. 3B), suggesting that HMGN2 modulates the cell cycle through the regulation of cyclin D1.

To determine the effect of HMGN2 on osteosarcoma cell invasion and metastasis, Transwell assay and wound-healing assay were carried out. As was shown in Fig. 4A, the migrative ability of SaO2 and U-2 OS cells in the HMGN2 group was lower than that in the CON and GFP groups. However, there were no significant differences between the CON and GFP groups. Furthermore, a Transwell assay was performed to determine the ability of cells to invade a matrix barrier. The representative micrographs of Transwell filters are shown in Fig. 4B. The invasive cell count demonstrated that invasive potential was significantly reduced in the HMGN2 group relative to the CON and GFP groups.

The action mechanism of many anticancer factors is based on their ability to induce apoptosis. Consequently, SaO2 and U-2 OS osteosarcoma cells treated with HMGN2 lentivirus underwent apoptosis as their mode of cell death. At the end of the incubation period for 24, 48 and 72 h, the osteosarcoma cells were stained using an Annexin V-FITC/PI detection kit. As shown in Fig. 4C, the number of SaO2 and U-2 OS apoptotic cells in the HMGN2 group significantly increased compared with that in the GFP and CON groups at various time points. To further investigate whether HMGN2 affected the sensitivity to chemotherapy, the apoptosis in HMGN2-overexpressed osteosarcoma cells was induced by chemotherapy. A significant increase of apoptosis incidence was detected in cells treated with Dox, Cis, and Mtx for 24 h (Fig. 4D). Overexpression of HMGN2 resulted in a further increase of apoptosis induced by these anticancer agents, suggesting that HMGN2 increased the sensitivity to chemotherapy.

The in vitro experiments confirmed that HMGN2 efficiently inhibited the growth and migration of U2-OS and SaO2 cells. However, whether HMGN2 has the same inhibitory effect on in vivo osteosarcoma remains to be determined. We investigated the antitumor effect of HMGN2 in vivo using SaO2 and U2-OS xenograft models. The mean volumes of SaO2 and U2-OS xenograft tumors were 52.3±10.5 and 35.5±8.6 mm³ in the experimental mice prior to treatment. On day 14, the average volumes of SaO2 and U2-OS xenograft tumors were significantly smaller in the HMGN2 group than those in the CON and GFP groups (Fig. 5A). During the whole tumor growth period, the tumor growth activity was measured. Tumors treated with HMGN2 lentivirus grew substantially slower than the GFP group (Fig. 5B). When the tumors were harvested, the average weights of SaO2 and U-2 OS xenograft tumors in HMGN2 group were significantly lighter than those in the GFP group (^**P<0.01; Fig. 5C). These results in vivo indicated that overexpression of HMGN2 was able to inhibit SaO2 and U-2 OS cell growth.