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Upregulation of CENPF is linked to aggressive features of osteosarcoma

Centromere protein F (CENPF) plays a key role in the regulation of the cell cycle. The present study revealed that CENPF was overexpressed in a variety of tumors and associated with the poor prognosis of osteosarcoma. The mRNA expression levels of CENPF were analyzed using the Gene Expression Profiling Interactive Analysis database and the protein levels of CENPF were detected in the specimens from patients with osteosarcoma using immunohistochemistry. Cell proliferation, cell cycle and flow cytometry assays were performed after the transfection of control or CENPF plasmids into osteosarcoma cells. A xenografts assay was used to determine the effects of CENPF on tumor growth in vivo. The results showed that CENPF was upregulated in osteosarcoma tissues and associated with high-grade tumor stage (P=0.023) and intraglandular dissemination (P=0.046). The transfection-induced depletion of CENPF in human osteosarcoma MG-63 and U-2 OS cell lines inhibited cell proliferation, stimulated apoptosis and induced cell cycle arrest. Induced CENPF depletion in MG-63 cells inhibited tumor growth of osteosarcoma cells in mice. These findings suggested that elevated CENPF levels contributed to increased cell proliferation by mediating apoptosis and cell cycle in osteosarcoma. Therefore, CENPF might be a potential biomarker for poor prognosis of osteosarcoma.


Introduction
Osteosarcoma is a type of primary malignant tumor that originates in the bones (1,2). Each year in the United States 800-900 new cases are diagnosed of which ~400 occur in children and adolescents under 20-years of age recent decades years (3). Therefore, osteosarcoma has become the most common primary malignant bone tumor in children and adolescents (4). Due to the combination of surgery and chemotherapy, the survival rate of osteosarcoma has been significantly improved with a current overall expected cure rate of 50-65% (5).
Centromere protein F (CENPF) encodes a protein that binds with the centromere-kinetochore complex, and is located on the human chromosomal 1q41 (6). CENPF is a cell cycle-associated nuclear protein that is expressed at low levels during the G 0 /G 1 phase and congregates in the nuclear matrix during S phase, with the highest expression in G 2 /M phase (7). CENPF protein has a broad expression in multiple tissues, such as the testis, bone marrow and lymph node (8). CENPF is also highly expressed in several types of human tumors, such as breast cancer, pancreatic carcinoma and prostate cancer and has been identified as a protein marker for tumor cell proliferation (9,10). To the best of our knowledge, the role of CENPF remains unclear in osteosarcoma. The aim of the present study was to investigate the relationship between CENPF expression and prognosis of patients with osteosarcoma.

Materials and methods
Patients and tissues. From January 2016 to December 2018, 67 patients with osteosarcoma from the Jiangxi Cancer Hospital (Jiangxi, China) were included in the present retrospective study. All patients had a complete medical history and underwent a physical pre-operative examination and primary tumor resection at hospital. The inclusion criteria were: i) Complete medical history; and ii) Physical pre-operative examination diagnosed by pathology. Patients who received preoperative radiotherapy or chemotherapy were excluded. The surgically removed tumor and adjacent normal tissue (5 mm distance from the tumor margin) was immediately fixed with 4% formalin at room temperature for 48 h and the diagnosis of osteosarcoma confirmed by two independent pathologists. The patients provided written consent for postoperative specimens to be used for scientific research before the operation. All experiments were approved by The Human Ethics Committee of Jiangxi Cancer Hospital.
Pathological evaluation. All clinical and clinicopathological characteristics were collected from the medical record of the patients and the staging of tumors were performed according to The American Joint Committee on Cancer (11). The expression levels of CENPF in osteosarcoma were determined by two independent pathologists.
IHC analysis. The tumor tissues and adjacent tissues were cut into ~3-mm thick sections and fixed in 4% formalin for 24 h at room temperature. Subsequently, 4-µm sections were cut from the blocks, baked at 70˚C for 30 min, dewaxed with dimethylbenzene for 30 min and rehydrated with a decreasing gradient series (100, 95, 85 and 75%) of ethanol for 30 min. To antigen retrieve, slides were heated in the citrate buffer at 100˚C (pH 6.0) for 20 min in a microwave oven. To avoid interference with endogenous peroxidase, the slides were immersed in 3% hydrogen peroxide for 5 min at room temperature. Tissues were blocked using 10% goat serum (cat. no. E510009; BBI Life Sciences Corp.) for 20 min at room temperature, then incubated with anti-CENPF antibody (1:200 dilution; cat. no. ab224813; Abcam) at 4˚C overnight. After washing with PBS three times, slides were incubated with horseradish peroxidase-labeled secondary antibody at room temperature for 30 min (1:1,000; goat anti-rabbit IgG; cat. no. ab6721; Abcam). Thereafter, slides were stained using 3,3-diaminobenzidine reagent for 2 min at room temperature, followed by counterstaining with hematoxylin for 1 min at room temperature. Finally, all sections were sealed with neutral balata and images were captured under a light microscope. The H-score system was used to evaluate the IHC score. Patients were divided into high (H-score ≥200) or low CENPF expression groups (H-score <200). Cell transfection and the construction of stable cell lines. The short hairpin (sh)RNA of lentivirus vector (5'-AAA ATT CAA GAG CTT GAA GGA CA-3') and scrambled vector (non-targeting sequence, 5'-AGG TTA AGT CGC CCT CGC TCG AG-3') were assembled by Shanghai GenePharma Co., Ltd. The lentivirus vectors (C06002; Shanghai GenePharma Co., Ltd.; 3rd generation) were transfected into 293T cells using X-tremeGENE HP DNA (cat. no. 6366236001; Roche Diagnostics). The supernatant was harvested 48 h post-transfection to concentrate lentivirus according to the instructions. pMDL:VSVG:pRSV-Rev: Sh-vector were transfected in a ratio of=5:3:2:5 in 100 mm Cell culture dishes, the optical density (OD) 260/280 of all plasmid =1.8-1.9.
On the third day when MG-63 and U-2 OS were infected with lentivirus, MOI=1:10, 2 µg/ml of puromycin (cat. no. P8230; Beijing Solarbio Science & Technology Co., Ltd.) was added to screen positive cells for 1 week and the drug concentration was maintained at all times.
Immunoblotting. All protein was obtained from cells, including wildtype and transfected MG-63 and U2-OS cells using RIPA buffer (cat. no. R0010; Beijing Solarbio Science & Technology Co., Ltd.) according to the manufacturer's protocol. Protein concentration was determined using the Bradford Protein Assay kit (cat. no. PC0010; Beijing Solarbio Science & Technology Co., Ltd.). Total protein (30 µg/lane) was separated by 10% SDS-PAGE, transferred onto a PVDF membrane, and blocked with 5% fat-free milk in TBST buffer (TBS buffer with 0.5% Tween-20) for 1 h at room temperature. Then, the membranes were incubated with anti-CENPF antibody (1:1,000 dilution; cat. no. ab224813; Abcam) overnight at 4˚C. After washing with TBST buffer, the PVDF was incubated with goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (1:5,000 dilution; cat. no. ab6721; Abcam) for 1 h at room temperature. Subsequently, the bands were visualized using an ECL kit (cat. no. 32132; Thermo Fisher Scientific, Inc.) and ImageJ software v.1.8 (National Institutes of Health).
Colony formation assay. MG-63 and U-2 OS cells were stably transfected with control or CENPF shRNA lentivirus, digested and seeded into six-well plates (10 3 cells/well) for 10 days. Then, cell colonies of cells were fixed with 4% paraformaldehyde for 30 min at 4˚C and stained using crystal violet (0.2%) for 30 min at room temperature. The number of cell colonies were counted using a light microscope (IX73; Olympus Corporation) and ImageJ software v.1.8.0 (National Institutes of Health).

Cell Counting Kit (CCK)-8 assay.
To detect cell proliferation, osteosarcoma cells were seeded into 96-well plates in five replicates at a concentration of 10 3 cells/well with 200 µl complete medium and incubated for 5 days. Cell proliferation was determined using CCK-8 reagent (cat. no. E606335; BBI Solutions). Simply, 20 µl of CCK-8 was added into each well, incubated for 2 h at 37˚C, and the absorbance was measured at 450 nm using a microplate reader.
Flow cytometry assays. Both cell cycle and apoptosis analysis were performed using a flow cytometer. For cell cycle assay, control and CENPF-knockdown cells were digested with 0.25% trypsin for 3 min at 37˚C and washed with PBS buffer. For cell cycle analysis, cells were fixed with 70% ethanol for 30 min at room temperature and stained with propidium iodide (PI; cat. no. 421301; BioLegend, Inc.) in the presence of RNase A and incubated for 20 min at room temperature. For the detection of early and late apoptosis, living cells were double stained with Annexin V-FITC and PI (CA1020; Beijing Solarbio Science & Technology Co., Ltd.) for 15 min at room temperature. Subsequently, osteosarcoma cells were analyzed using a flow cytometer (FC500; BD Biosciences) and FlowJo software v.7.6 (FlowJo LLC).
Animal models. A total of six female nude-BALB/c mice (seven-weeks-old, 18-22 g) were obtained from Chengdu Feike Biotechnology Co., Ltd. and maintained in a SPF environment with 12 h light/dark cycle, 22±2˚C and air humidity, 55±10%. All mice were provided with food and water ad libitum. Human endpoints were monitored every day, such as body weight loss of 20% and tumor diameter >20 mm. All mice were randomly divided into two groups: Subcutaneously inoculated with 10 6 control MG-63 cells or subcutaneously inoculated with 10 6 CNEPF stably-depleted MG-63 cells. Tumor sizes were measured each 3 days. After 30 days, all mice were sacrificed and the tumors were isolated. Tumors were frozen immediately, followed by total protein extraction. The tumor volume was calculated using: V = (Length x width 2 )/2. The average tumor volume was 180 mm 3 . Euthanasia was performed using intraperitoneal sodium pentobarbital 100 mg/kg injection. Euthanasia was confirmed by cervical dislocation. All experiments were approved by The Laboratory Animal Ethics Committee of Jiangxi Cancer Hospital (Jiangxi, China; approval. no. SYXK 2019-0522).
Statistical analysis. Data were derived from three independent biological replicates. Differences between two groups were tested using two-tailed, unpaired Student's t-tests. The relationships between CENPF and clinicopathological characteristics of 67 patients with osteosarcoma were analyzed using Pearson χ 2 test or Yates continuity corrected χ 2 test. The data was displayed by GraphPad Prism v5 (GraphPad Software, Inc.). Data are presented as mean ± standard deviation (unless otherwise shown). P<0.05 was considered to indicate a statistically significant difference.

Results
High expression levels of CENPF mRNA are associated with poor OS and DFS rates in patients with osteosarcoma from the GEPIA database. A total of 262 osteosarcoma tissues and two normal tissues from healthy patients without osteosarcoma were included from the GEPIA database and analyzed. The resultant expression boxplot demonstrated that CENPF was significantly overexpressed in osteosarcoma specimens at the mRNA level (Fig. 1A). The Kaplan-Meier showed that high expression levels of CENPF are associated with poor OS and DFS rates in patients with osteosarcoma (P=0.012 and 0.0033, respectively; Fig. 1B). These results indicated that CENPF was upregulated in osteosarcoma and associated with poor prognosis. High expression levels of CENPF protein are associated with high T stage and intraglandular dissemination. A total of 67 patients with osteosarcoma were collected from 2016 to 2018. The protein expression levels of CENPF protein were evaluated using IHC, and the clinicopathological information of the patients was analyzed. As shown in the Fig. 2A-C, CENPF protein levels were significantly upregulated in osteosarcoma specimens compared with adjacent normal tissues. Performing clinicopathological characteristics analysis revealed that CENPF was upregulated in 82% (55 vs. 67) osteosarcoma specimens and high expression levels of CENPF protein were associated with T stage and intraglandular dissemination (Table I). These clinical data indicated high expression of CENPF was associated with poor prognosis in patients with osteosarcoma.

Depletion of CENPF in human osteosarcoma cell lines by CENPF shRNA transfection.
To understand the functions of CENPF in osteosarcoma, CENPF was depleted by shRNA transfection in human osteosarcoma cell lines MG-63 and U-2 OS. As shown in Fig. 3A, the mRNA expression level of CENPF was significantly reduced in both cell lines after the stable transfection of CENPF shRNA plasmids. Similarly, immunoblotting showed that the protein expression levels of   CENPF were decreased in shRNA stably transfected cells (Fig. 3B). Subsequent studies used CENPF-knockdown cell lines screened by puromycin.

Depletion of CENPF inhibits osteosarcoma tumor growth in vivo.
To determine whether CENPF inhibited tumor growth in vivo, control or CENPF stably-depleted MG-63 cells were implanted into nude-BALB/c mice, and the tumor volume was quantified every 3 days. The tumor growth curve revealed that xenografts with CENPF depletion were significantly smaller compared with the control xenografts (Fig. 5A). Immunoblot assays showed that xenografts derived from CENPF stably-depleted tumor tissues had a lower expression level of CENPF compared with control tissues (Fig. 5B). Therefore, these data revealed that depletion of CENPF inhibited the growth osteosarcoma tumors in vivo.

Discussion
As a component of the centromere-kinetochore complex, CENPF has been reported to play a notable role in carcinogenesis and affects the progression of a number of human tumors, such as breast cancer, pancreatic carcinoma and prostate cancer (1,(14)(15)(16)(17)(18). Previous studies have reported that CENPF is localized in the nucleus of prostate cancer cells, and its expression is associated with the poor prognosis of patients with prostate cancer (19)(20)(21). By analyzing the data in TCGA database and the protein levels of CENPF in patient samples, the present study found that the mRNA and protein expression levels of CENPF were both significantly upregulated in osteosarcoma tissues. Patients with high CENPF expression levels had poorer OS and DFS rates. Similarly, Li et al (22) analyzed four mRNA microarrays from the Gene Expression Omnibus database and found that CENPF is overexpressed in lung cancer and associated with poor prognosis. CENPF has already been reported to be overexpressed in the early-stages of hepatocellular carcinoma (HCC) and as a tumor-associated antigen (23,24). Previous studies have also confirmed the  diagnostic value of CENPF in early HCC and CENPF was upregulated in HCC and was associated with poor outcome (25)(26)(27)(28).
In the present study, the IHC data of osteosarcoma samples demonstrated that CENPF upregulated in 82% of tumors, and was associated with T stage and intraglandular dissemination. In a previous study on prostate cancer, 8,066 out of 9,055 (89%) stains were found to be CENPF-positive, whereas normal prostate tissues showed absent or weak CENPF staining (29). In prostate cancer, high expression levels of CENPF are associated with indicators of advanced pathological stage, such as high Gleason grade and lymph node metastasis (19)(20)(21)29). Similarly, a previous study revealed that high CENPF expression levels are associated with a high Ki67-labeling index, showing that CENPF is associated with tumor cell proliferation (29). In line with the findings of the current study, CENPF is also upregulated in other human tumors, including breast cancer, esophageal cancer and nasopharyngeal cancer (8,(30)(31)(32)(33)(34)(35).
To further explore the effect of CENPF on osteosarcoma cells, the present study performed cell cloning using cells transfected with CENPF shRNA lentivirus, combined with flow cytometry and apoptosis analysis. CENPF was found to affect the cell cycle and apoptotic pathway and promoted the proliferation of tumor cells. The current study was consistent with previous reports demonstrating that CENPF promoted tumor cell proliferation. For example, Laoukili et al (36) identified CENPF as a direct target gene of forkhead box protein M1, which is a notable cell cycle regulator which activates MAPK and PI3K/AKT signaling pathways. Sun et al (10) reported that overexpression of CENPF is associated with P53 signaling pathways. Notably, the AKT/mTOR signaling pathway is inhibited when CENPF protein expression is depleted via transfection in breast cancer cells, and depletion of CENPF inhibits the synthesis and phosphorylation of AKT/mTOR pathway components in these cells, such as phosphorylated (p)-AKT and p-mTOR (10).
However, the limitation of the current study is that it is a retrospective study and the expression levels of CENPF was analyzed in a limited number of osteosarcoma specimens. Additionally, the present study did not involve detailed research into the mechanism of CENPF. Therefore, for clinical application, such as inhibited tumor by targeting CENPF using antisense oligonucleotide, a larger number of samples and in-depth exploration are needed.
In conclusion, the data from the present study indicated that CENPF was an independent negative prognostic factor in patients with osteosarcoma. CENPF promoted cell proliferation by the regulation of cell cycle and apoptosis. These data may therefore provide a possible biomarker and prognostic prediction factor of osteosarcoma.