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MAFG-driven osteosarcoma cell progression is inhibited by a novel miRNA miR-4660

Osteosarcoma (OS) is the most common primary bone malignancy in the adolescent population. MAFG (v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog G) forms a heterodimer with Nrf2 (NF-E2-related factor 2), binding to antioxidant response element (ARE), which is required for Nrf2 signaling activation. We found that MAFG mRNA and protein expression is significantly elevated in human OS tissues as well as in established and primary human OS cells. In human OS cells, MAGF silencing or knockout (KO) largely inhibited OS cell growth, proliferation, and migration, simultaneously inducing oxidative injury and apoptosis activation. Conversely, ectopic overexpression of MAFG augmented OS cell progression in vitro. MicroRNA-4660 (miR-4660) directly binds the 3′ untranslated region (UTR) of MAFG mRNA in the cytoplasm of OS cells. MAFG 3′ UTR luciferase activity and expression as well as OS cell growth were largely inhibited with forced miR-4660 overexpression but augmented with miR-4660 inhibition. In vivo, MAGF short hairpin RNA (shRNA) or forced overexpression of miR-4660 inhibited subcutaneous OS xenograft growth in severe combined immunodeficient mice. Furthermore, MAFG silencing or miR-4660 overexpression inhibited OS xenograft in situ growth in proximal tibia of the nude mice. In summary, MAFG overexpression-driven OS cell progression is inhibited by miR-4660. The miR-4660-MAFG axis could be novel therapeutic target for human OS.


INTRODUCTION
Osteosarcoma (OS) is the most common histological form of primary bone cancer in adolescents and children. 1 It ranks the eighth-mostcommon form of childhood cancer, comprising around 2.4% of all cancers in pediatric patients and around one-fifth of all primary bone malignancies. 2,3 Advanced OS is characterized by high malignancy grade, dismal prognosis, early local recurrence, and distant metastasis. 2,3 OS patients can develop micro-metastases in the lung with dismally low 5-year survival rates. 4 The standard treatment options for OS patients include surgery combined with chemotherapy. 5,6 The large cooperative group studies have implied that there has been little to no further improvement of overall survival for OS patients in the past years. 6 To improve clinical outcome, there is an urgent need to explore the pathological mechanisms of OS tumorigenesis and progression. 7,8 Recent molecular genetic studies of OS have changed our view on the disease. 5,9 The molecularly targeted therapies are the current research focus of OS. 2,5,9 MAFG (v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog G) is a small MAF protein belonging to the basic leucine zipper (bZIP) family of transcription factors. 10 The MAFG bZIP structure has a basic DNA binding region and a leucine zipper structure 11 but no canonical transcriptional activation domain. 11 MAFG is broadly expressed in all human tissues but is relatively abundant in lung, lymph node, skeletal muscle, and thyroid tissues. 12 MAFG knockout (KO) mice presented with a mild neuronal phenotype and mild thrombocytopenia. 13 MAFG forms heterodimers with Nrf2 (NF-E2-related factor 2) protein, the latter being an essential regulator of antioxidant genes and detoxification enzymes. 14,15 MAFG-Nrf2 heterodimers bind and activate the antioxidant response element (ARE) in the promoter regions of many genes involved in antioxidant defense. 16 These genes including heme oxygenase 1 (HMOX-1), NAD(P)H quinone oxidoreductase 1 (NQO1), glutamate cysteine ligase catalytic subunit (GCLC), and many others. 14,15 MAFG KO mouse embryonic fibroblasts (MEFs) failed to induce the Nrf2 signaling cascade in response to stress. 10 In this study, we examine the expression and potential functions of MAFG in human OS. We show that MAFG overexpression is important for OS cell growth in vitro and in vivo.

MAFG overexpression in human OS
OS comprises almost 60% of all the common histological subtypes of bone sarcoma. 3 To examine MAFG expression in OS, we first consulted the TARGET Pan-Cancer (PANCAN) database to examine RNA sequencing (RNA-seq) data in children's sarcoma tissues and normal adjacent tissues via a UCSC Xena project. As shown, in children's sarcoma tissue (n = 1,524) MAFG mRNA expression is significantly upregulated (p < 0.05 versus normal tissues; n = 66) (Figure 1A). Furthermore, MAFG mRNA upregulation is detected in 14 cases of recurrent children's sarcoma samples ( Figure 1A). Importantly, the average survival of patients with MAFG-high sarcoma (RNA-seq R 4.210) is lower than those with MAFG-low (MAFG RNA-seq < 4.210) sarcoma (p = 0.029; Figure 1B). These results show that MAFG is upregulated in children's sarcoma tissues and is associated with poor overall survival.
To confirm the significance of the bioinformatics observations, we examined MAFG expression in human OS tissues. OS tumor tissues (T) and surrounding normal bone tissues (N), derived from a total of 12 primary OS patients, were analyzed. By quantitative real-time PCR, MAFG mRNA expression in OS tissues was 5.2-fold greater than in normal tissues (p < 0.05; Figure 1C). The mRNA expression of heme oxygenase-1 (HMOX1), a key Nrf2 response gene, 27,28 was also upregulated in OS tissues (p < 0.05 versus N tissues; Figure 1D). Western blotting studies were performed to test protein expression and showed that MAFG and HMOX1 protein expression was elevated in human OS tissues ( Figure 1E). Quantification analyses integrating blotting data from 12 sets of human tissues confirmed that MAFG and HMOX1 protein upregulation was significant (p < 0.05 versus N tissues; Figures 1F and 1G).
We also examined MAFG expression in established OS cell lines (U2OS and MG63) and primary human OS cells. The primary OS cells were derived from three primary OS patients, namely pOS-1, pOS-2, and pOS-3 (see Materials and methods). As shown MAFG-HMOX1 mRNA expression is relatively low in OB-6 human osteoblastic cells and primary human osteoblasts ( Figures 1H and 1I). In the established and primary human OS cells, MAFG-HMOX1 mRNA upregulation was detected ( Figures 1H and 1I). Western blotting results ( Figure 1J) confirmed MAFG-HMOX1 protein overexpression in established (U2OS and MG63) and primary OS cells, whereas low expression is detected in primary human osteoblasts. Together, these results confirmed MAFG overexpression in human OS.
The potential pro-cancerous function of MAFG was tested in additional human OS cells. In primary human OS cells derived from two other OS patients, pOS-2 and pOS-3, as well as in established OS cell lines (U2OS and MG63), the application of the MAFG-shRNA lentivirus led to an 80%-90% reduction of MAFG mRNA ( Figure 2I; p < 0.05 versus shC cells). Functional studies demonstrated that MAFG shRNA in OS cells potently inhibited cell viability (CCK-8 OD; Figure 2J; p < 0.05 versus shC cells), cell proliferation (nuclear EdU ratio; Figure 2K; p < 0.05 versus shC cells), and migration (Figure 2L; p < 0.05 versus shC cells). Collectively, these results show that MAFG silencing or KO inhibited cell viability, growth, proliferation, and migration in established and primary human OS cells.
In OS cells, proliferation arrest and/or oxidative injury is anticipated to result in cell apoptosis. Testing the potential effect of MAFG depletion on cell apoptosis, we found that the ratio of terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL)-positive nuclei was significantly increased in pOS-1 cells-bearing the MAFG-shRNA or MAGF-KO construct ( Figure 3E; p < 0.05 versus shC+Cas9 cells). Annexin V FACS assay results ( Figure 3F) further confirmed that MAFG silencing or KO caused apoptosis activation. Significant caspase-3 activation was also detected in pOS-1 cells with MAFG silencing or KO ( Figure 3G). The two other MAFG shRNAs, sh-MAFG-Seq1/2, induced ROS production (CellROX intensity increase; Figure S1F), mitochondrial depolarization (JC-1 green monomer intensity increase; Figure S1G), and apoptosis activation (nuclear TUNEL ratio increase; Figure S1H) in pOS-1 cells. These results confirm that MAFG silencing/depletion induces robust apoptosis activation in primary OS cells.
To test whether oxidative stress is the primary cause of cell apoptosis in MAFG-depleted cells, we applied several known antioxidants, including N-acetylcysteine (NAC), pyrrolidine dithiocarbamate (PDTC), 30,31 and AGI-1067. 32,33 As shown, in pOS-1 cells MAFG shRNA-and MAFG KO-induced viability (CCK-8 OD) reduction,  proliferation inhibition (nuclear EdU ratio reduction), and cell apoptosis (increased ratio in TUNEL-positive nuclei) were largely attenuated by the ROS scavengers ( Figure 3H). These results indicate MAFG silencing/KO resulted in ROS accumulation to induce OS cell apoptosis.

Ectopic MAFG overexpression further promotes OS cell progression in vitro
Based on the results, we hypothesized that MAFG overexpression would promote OS cell progression in vitro. A MAFG-expressing GV369 vector was transduced into pOS-1 cells, and stable cells were established (OE-MAFG cells). Quantitative real-time PCR assay results ( Figure 4A) confirmed that MAFG mRNA levels were increased over seven-fold in the OE-MAFG cells, and MAFG protein expression was significantly elevated ( Figure 4B). Further, HMOX1 mRNA and protein expression was increased ( Figures 4B and 4C).
These results indicate a high percentage of direct binding between the two. 34 By employing an RNA-immunoprecipitation (RNA-IP) assay, we demonstrated that endogenous MAFG mRNA and miR-4660 were pulled down by an anti-Ago2 antibody in pOS-1 cells ( Figure 5B). The non-specific anti-immunoglobulin G (IgG) antibody failed to pull down MAFG mRNA and miR-4660 ( Figure 5B). RNA pulldown assay results further showed that biotinylated-miR-4660 directly associated with MAFG mRNA, but not with two other MAF family genes, MAFF mRNA and MAFK mRNA ( Figure 5C). In addition, fluorescence in situ hybridization (FISH) assay results demonstrated that miR-4660 (in red) and MAFG mRNA (in green) were co-localized mainly in the cytoplasm of pOS-1 cells (Figure 5D). These results suggested a high probability for the two to bind. 34 Based on the above results, we hypothesized that ectopic miR-4660 overexpression would alter MAFG expression. A lentiviral construct encoding pre-miRNA-4600 (lv-pre-miR-4660) was transduced into pOS-1 cells, and two stable cell lines, lv-pre-miR-4660-L1 and lvpre-miR-4660-L2, were established via puromycin selection. The quantitative real-time PCR results ( Figure 5E) confirmed that the expression of mature miRNA-4660 increased over 20-fold in lvpre-miR-4660 cells (p < 0.05 versus cells with miRNA control construct/lv-miRC). A dual-luciferase reporter assay demonstrated that MAFG 3 0 UTR luciferase reporter activity was significantly decreased after the forced miR-4660 overexpression in pOS-1 cells (p < 0.05 versus lv-miRC cells) ( Figure 5F). Consequently, MAFG mRNA ( Figure 5G) and protein ( Figure 5H Figure 5J). RNA-IP experiment results confirmed that only WT-miR-4660 immunoprecipitated with MAFG mRNA, while the mutant failed ( Figure 5K). Transfection the WT-miR-4660, but not the Mut-miR-4660, decreased MAFG 3 0 UTR luciferase reporter activity ( Figure 5L) as well as expression of MAFG mRNA ( Figure 5M) in pOS-1 cells.
We also analyzed expression of miR-4660 in the human OS tissues described in Figure 1. The quantitative real-time PCR assay results ( Figure 5P) demonstrated that miR-4660 expression in OS tumor tissues (T) was significantly lower than that in the surrounding normal tissue (N). Furthermore, low miR-4660 expression was detected in primary OS cells (pOS-1, pOS-2, and pOS-3) and established OS cell lines (U2OS and MG63) ( Figure 5Q), compared to expression in OB-6 osteoblastic cells and primary human osteoblasts ( Figure 5Q). Therefore, in human OS tissues, low miR-4660 expression is correlated with MAFG elevation (see Figure 1).

MAFG silencing or miR-4660 overexpression inhibits growth of subcutaneous OS xenografts and in situ OS xenografts in mice
To study the potential role of the miR-4660-MAFG axis on OS cell growth in vivo, a xenograft mouse model was applied. The pOS-1 cells were subcutaneously (s.c.) injected into the flanks of severe combined immunodeficient (SCID) mice. Within 3 weeks, OS xenografts were established, with each tumor close to 100 mm 3 . Mice were then randomly assigned into three groups (day 0), with intratumoral injection of MAFG shRNA lentivirus (MAFG shRNA; see Figure 2), pre-miR-4660 lentivirus (lv-pre-miR-4660; see Figure 5), or the control virus (miRC+shC). The tumor growth curve results confirmed that tumors bearing MAFG shRNA or lv-pre-miR-4660 grew significantly slower than the control tumors ( Figure 8A). The estimated daily tumor growth, calculated by the formulation (tumor volume at day 42 À tumor volume at day 0)/42, confirmed that MAFG shRNA or miR-4660 overexpression robustly inhibited pOS-1 xenograft growth in SCID mice ( Figure 8B). At day 42, tumors of all three groups were isolated and individually weighed. Results showed that xenografts bearing MAFG shRNA or lv-pre-miR-4660 were significantly lighter than the control xenografts ( Figure 8C). Mouse body weights were not significantly different between the three groups ( Figure 8D), and no significant toxicity was observed.
To further support a role of the miR-4660-MAFG axis in OS cell growth in vivo, genetically modified primary OS cells were injected into the proximal tibia of nude mice to establish an animal model of in situ OS. These cells included stable pOS-1 cells expressing MAFG shRNA lentivirus (MAFG shRNA), pre-miR-4660 lentivirus (lv-pre-miR-4660), or the control virus (miRC+shC). Results demonstrated that, 24 days (day 24) after the cell injection, the volumes of in situ pOS-1 xenografts bearing MAFG shRNA or lv-pre-miR-4660 were significantly lower than xenografts with control virus (Figures 8H   and 8I). At day 24, the in situ pOS-1 xenografts were isolated and homogenized. As demonstrated, MAFG mRNA levels were significantly decreased in in situ pOS-1 xenograft tissues with MAFG shRNA and lv-pre-miR-4660 ( Figure 8J). The mature miR-4660 levels were increased over eight-fold in lv-pre-miR-4660-expressing xenograft tissue ( Figure 8K). These results demonstrate that MAFG shRNA or forced miR-4660 overexpression potently inhibits OS xenografts in situ growth in mice.

DISCUSSION
MAFG forms a heterodimer with Nrf2 to bind ARE, initiating transcription of detoxification enzymes and antioxidant genes. [35][36][37] Recent studies suggest that MAFG could play an important role in cancer progression. 38 The overexpression of MAFG in hepatocellular carcinoma . miR-4660-induced anti-OS cell activity is due to MAFG silencing Stable pOS-1 cells with the pre-miRNA-4600-expressing construct (lv-pre-miR-4660) were further transduced with or without a lentiviral 3 0 UTR null MAFG expression vector: MAFG (UTR null). Control cells were with scramble control microRNA (lv-miRC). Expression of listed genes was tested by quantitative real-time PCR and western blotting assays (A-D). Cells were further cultured for applied time periods; cell proliferation, ROS production, and apoptosis were tested by EdU incorporation (E), CellROX intensity (F), and TUNEL staining (G) assays, respectively, with results quantified. Stable pOS-1 cells with the lenti-CRISPR-Cas9-MAFG-KO construct (ko-MAFG) were transduced with lv-pre-miR-4660, lv-antagomiR-4660, or lv-miRC; expression of mature miR-4660 is shown (H). Cells were further cultured for applied time periods; cell proliferation (I), ROS production (J), and apoptosis (K) were tested similarly. Pare, parental control cells. Data were presented as mean ± standard deviation (SD, n = 5). *p < 0.05 versus Pare cells (H-K). Experiments in this figure were repeated five times with similar results obtained.
is associated with tumor progression and decreased survival. 38 In nonsmall cell lung cancer, MAFG inhibition or downregulation induced ROS production, sensitizing cancer cells to cisplatin-induced apoptosis. 39 BRAF V600E -stabilized MAFG initiated recruitment of a co-repressor complex to CpG island methylator phenotype (CIMP) gene promoters in colorectal cancer cells, which is associated with tumorigenesis and cancer growth. 40 Silencing of MAFG potently inhibited colorectal cancer cell growth. We provide evidence to show that MAFG is a novel oncogenic gene and potential therapeutic target of OS. MAFG mRNA and protein expression are significantly elevated in human OS tissues, compared to low expression in normal bone tissues and osteoblasts. In primary and established OS cells, MAGF shRNA or KO inhibited OS cell growth, proliferation, and migration, while simultaneously eliciting cell-cycle arrest and cell apoptosis. Conversely, ectopic overexpression of MAFG further promoted OS cell progression in vitro. Significantly, MAFG silencing potently inhibited growth of subcutaneous OS xenografts and in situ OS xenografts in mice.
Bai et al. 41 have reported that low levels of ROS, detected in human OS tissues and cells, are important for OS cell proliferation and invasion. However, sustained ROS production and profound oxidative injury induced OS cell growth arrest and apoptosis. Wang et al. 42 have shown that andrographolide induced OS cell apoptosis via ROS production and downstream JNK activation. Similarly, hyperthermia-induced OS cell apoptosis was associated with ROS production. 43 Ionizing radiation (IR) was also found to induce robust ROS production to promote U2OS cell apoptosis. 44 Such action by IR was attenuated by Nrf2 upregulation-induced antioxidant response. 44 We found that MAFG silencing results in Nrf2-signaling inhibition, leading to significant ROS production and oxidative injury. Antioxidants, including NAD, PDTC, and AGI-1067, inhibited MAFG silencing-induced OS cell apoptosis. miRNA binds the 3 0 UTR of the complementary mRNAs, causing targeted mRNA translation inhibition and/or mRNA degradation. 25,26 miRNA is often dysregulation in human OS [22][23][24] and is associated with tumorigenesis progression. 25,26 The potential function of miR-4660 is largely unknown. A recent study by Tu et al. 45 has demonstrated that miR-4660 is downregulated in patients with oxalosis. It bound directly to 3 0 UTR of alanine-glyoxylate aminotransferase (AGXT) to inhibit its expression. 45 The results of this study demonstrate that miR-4660 is a novel MAGF-targeting miRNA in OS cells. miR-4660 is localized in the cytoplasm of OS cells, where it can bind directly to 3 0 UTR of MAFG mRNA. In OS cells, ectopic overexpression of miR-4660 inhibited MAFG 3 0 UTR luciferase activity and expression. Both were, however, augmented with miR-4660 inhibition. miR-4660 overexpression mimicked MAFG silencing-induced actions, inhibiting OS cell proliferation and migration and inducing ROS production and apoptosis activation. Importantly, restoring MAFG expression completely reversed miR-4660 overexpressioninduced OS cell inhibition. Furthermore, exogenously altering miR-4660 expression failed to affect the function of MAFG-KO OS cells.
Significantly, decreased miR-4660 expression in human OS tissues correlates with MAFG upregulation.
OS accounts for 60% of the common histological subtypes of bone sarcoma. It is the primary malignant bone tumor that commonly affects adolescents and young adults. 6,46 Current chemotherapy for human OS consists of the combination of methotrexate, doxorubicin, and cisplatin (MAP). 2,6,47 The application of molecularly targeted therapies has so far failed to significantly improve the overall survival of advanced OS patients. Hence, further exploration of the pathology of OS is essential for improving the prognosis of patients with advanced OS. 2,6,47 Our results indicate that dysregulation of the miR-4660-MAFG axis is involved in the progression of OS. Targeting the miR-4660-MAFG axis could be a promising therapeutic strategy for this devastating malignancy.

Cell culture
Established human OS cell lines, U2OS and MG63, were obtained from the Cell Bank of Shanghai Institute of Biological Science (Shanghai, China). Cells were cultured using a previously described protocol. 48 The primary human OS cells derived from three human OS patients, pOS-1, pOS-2, and pOS-3, were provided by Dr. Ji at Nanjing Medical University, 49 and cells were cultured under the described conditions. 49,50 Primary OS cells at passages 3-10 were utilized. OB-6 human osteoblastic cells and primary human osteoblasts were provided again by Dr. Ji at Nanjing Medical University, cultured as previously described. 51,52 The protocols of the study were approved by the institutional animal care and use committee (IACUC) and ethics committee of Soochow University.

Human OS tissues
Human OS tissues and the matched surrounding normal bone tissues were from a set of 12 OS patients with written informed consent administrated at the Affiliated Children Hospital of Soochow University (Suzhou, China). Tissues were incubated with the tissue lysis buffer 53 and stored in liquid nitrogen. The protocols of using human tissues were approved by the ethics committee of Soochow University.

Quantitative real-time PCR
TRIzol reagents were applied to achieve total RNA, which was quantified and reversely transcribed. 54 The quantitative real-time PCR was performed by the SYBR Premix Ex TaqTM kit under the ABI-7500 PCR system (Shanghai, China). 54 mRNA expression was quantified by 2 ÀDDCt protocol with glyceraldehyde-3-phosphatedehydrogenase (GAPDH) as the internal control. Mature miR-4660 expression was detected using the TaqMan microRNA assay of hsa-miR-4660 (Applied Biosystems, Shanghai, China). The TaqMan MicroRNA Reverse Transcription kit (Applied Biosystems) was used for RNA (10 ng) reverse transcription via the stem-loop primer. U6 RNA was tested as the internal control for miR-4660. mRNA primers were listed in Table 1. mRNA primers of human GCLC, NQO1, and GAPDH were described previously. 55

RNA-IP
Cells were first incubated with the complete RIP lysis buffer (Beyotime). For each treatment, 1,000 mg total cell lysates were incubated with magnetic beads conjugated with anti-Argonaute 2 (Ago2, Santa Cruz Biotechnology) antibody or an anti-IgG antibody (Santa Cruz Biotechnology) overnight. Beads were washed and incubated with Proteinase K (Sigma). Finally, quantitative real-time PCR was performed to test the purified RNAs; MAFG mRNA and miR-4660 levels were normalized to input controls.

Western blotting
Western blotting assays were performed by well-established protocols as in our previous studies. 48,62 The same set of lysates was run separately in sister gels when testing different proteins. The band intensity was quantized by densitometric analysis using NIH ImageJ software. www.moleculartherapy.org

Cell viability
Cell viability was assessed through a CCK-8 assay. Briefly, cells with the applied genetic modifications were seeded into the 96-well tissue culture plates (at 3 Â 10 3 cells per well). Following incubation for 96 h, cell viability was estimated by recording the CCK-8 OD at 450 nm using a microplate reader.

EdU staining
OS cells with the applied genetic modifications were seeded into sixwell plates (at 1 Â 10 5 cells in each well) and cultured for 72 h. An EdU Apollo-567 assay kit (RiboBio, Guangzhou, China) was employed to quantitatively measure cell proliferation. The cell nuclei were stained with both EdU and DAPI, visualized under a fluorescent microscope (Leica, DM 4000, Germany). Cells in each field of view were then counted and analyzed. The nuclear EdU ratio, % versus DAPI, from at least 1,000 cells in five random views per treatment was calculated.

In vitro cell migration assay
On the upper Transwell chambers (8-mm pore, Corning, New York, NY, USA) OS cells (10,000 cells per chamber, in serum-free medium) with applied genetic modifications were plated, 63 with the lower chambers filled with 10% FBS complete medium. After 16 h, OS cells migrating to the lower chambers were fixed, stained, and counted.

Apoptosis detection
OS cells with the applied genetic modifications were seeded into sixwell plates (at 1 Â 10 5 cells in each well) and cultured. The detailed protocols for cell apoptosis assays, including nuclear TUNEL staining, Annexin V FACS, and caspase-3 activity assay, were described in the previous studies. 64,65 ROS detection OS cells with applied genetic treatments were seeded into the six-well plates at 1 Â 10 5 cells per well and stained with CellROX fluorescence dye (Thermo Fisher Scientific, Shanghai, China) for 1 h in the dark. The CellROX fluorescence intensity (at 625 nm) was detected and the representative CellROX images were presented as well.
JC-1 assay OS cells were seeded into the six-well plates and stained with JC-1 fluorescence dye for 30 min. JC-1 intensity was quantified via a fluorescence spectrofluorometer (F-7000, Hitachi, Japan) at test-wavelength of 488 nm (green). The representative JC-1 images, integrating both green fluorescence (at 488 nm) and red fluorescence (at 625 nm) channels, were presented as well.

ARE reporter assay
The primary human OS cells, pOS-1, were initially seeded in six-well plates at 50%-60% confluence. Cells were then transfected with the ARE-inducible firefly luciferase vector (from Dr. Jiang at Nanjing Medical University 57 ). Following the applied genetic treatment, total cell lysates (30 mg per treatment) were subjected to ARE-reporter luciferase activity assay under a luminescence machine.

Mice xenograft assay
The CB.17 female SCID mice and the nude mice were maintained under the Animal Facility of Soochow University (Suzhou, China). For each mouse, 5 Â 10 6 OS cells (in Matrigel-containing medium, no serum) were s.c. injected to the flanks. The subcutaneous OS xenografts were established within 3 weeks with each tumor close to 100 mm 3 in volume. Tumor-bearing mice were randomly divided into three groups and were treated as described. Mice body weight and bidimensional tumor measurements were recorded every 7 days. Tumor volume was estimated using the standard formula: (length Â width2)/2. For the in situ OS model, pOS-1 cells (3 Â 10 6 cells per mouse) with applied genetic modifications were injected to the proximal tibia of the nude mice. Twenty-four days after cell injection, in situ tumors were visualized under X-ray film. Tumors were isolated and measured and tumor tissues tested by quantitative real-time PCR analyses. The animal protocols were approved by IACUC and Ethic Committee of Soochow University.

Statistical analysis
Statistical analysis was performed with SPSS version 23.0 (SPSS, Chicago, IL, USA). All quantitative data were presented as the mean ± standard deviation (SD). Differences between two groups were compared by the Student's t test when they had a normal distribution. A one-way analysis of variance (ANOVA) followed by a Scheffé and Tukey test was used to compare data among groups when they had a normal distribution and homogeneous variances. p values less than 0.05 were considered statistically significant.