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Upregulation of CENPF Is Linked to Aggressive Features of Osteosarcoma

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Upregulation of CENPF Is Linked to Aggressive Features of Osteosarcoma ONCOLOGY LETTERS 22: 648, 2021 Upregulation of CENPF is linked to aggressive features of osteosarcoma PING‑AN ZOU, ZHENG‑XU YANG, XI WANG and ZHI‑WEI TAO Department of Bone and Soft Tissue Oncology, Jiangxi Cancer Hospital, Nanchang, Jiangxi 330029, P.R. China Received July 15, 2020; Accepted April 20, 2021 DOI: 10.3892/ol.2021.12909 Abstract. Centromere protein F (CENPF) plays a key role in 800‑900 new cases are diagnosed of which ~400 occur in the regulation of the cell cycle. The present study revealed that children and adolescents under 20‑years of age recent decades CENPF was overexpressed in a variety of tumors and associ‑ years (3). Therefore, osteosarcoma has become the most common ated with the poor prognosis of osteosarcoma. The mRNA primary malignant bone tumor in children and adolescents (4). expression levels of CENPF were analyzed using the Gene Due to the combination of surgery and chemotherapy, the Expression Profiling Interactive Analysis database and the survival rate of osteosarcoma has been significantly improved protein levels of CENPF were detected in the specimens from with a current overall expected cure rate of 50‑65% (5). patients with osteosarcoma using immunohistochemistry. Centromere protein F (CENPF) encodes a protein that Cell proliferation, cell cycle and flow cytometry assays were binds with the centromere‑kinetochore complex, and is performed after the transfection of control or CENPF plas‑ located on the human chromosomal 1q41 (6). CENPF is a cell mids into osteosarcoma cells. A xenografts assay was used to cycle‑associated nuclear protein that is expressed at low levels determine the effects of CENPF on tumor growth in vivo. The during the G0/G1 phase and congregates in the nuclear matrix results showed that CENPF was upregulated in osteosarcoma during S phase, with the highest expression in G2/M phase (7). tissues and associated with high‑grade tumor stage (P=0.023) CENPF protein has a broad expression in multiple tissues, and intraglandular dissemination (P=0.046). The transfec‑ such as the testis, bone marrow and lymph node (8). CENPF is tion‑induced depletion of CENPF in human osteosarcoma also highly expressed in several types of human tumors, such MG‑63 and U‑2 OS cell lines inhibited cell proliferation, as breast cancer, pancreatic carcinoma and prostate cancer and stimulated apoptosis and induced cell cycle arrest. Induced has been identified as a protein marker for tumor cell prolifera‑ CENPF depletion in MG‑63 cells inhibited tumor growth of tion (9,10). To the best of our knowledge, the role of CENPF osteosarcoma cells in mice. These findings suggested that remains unclear in osteosarcoma. The aim of the present study elevated CENPF levels contributed to increased cell prolifera‑ was to investigate the relationship between CENPF expression tion by mediating apoptosis and cell cycle in osteosarcoma. and prognosis of patients with osteosarcoma. Therefore, CENPF might be a potential biomarker for poor prognosis of osteosarcoma. Materials and methods Introduction Patients and tissues. From January 2016 to December 2018, 67 patients with osteosarcoma from the Jiangxi Cancer Osteosarcoma is a type of primary malignant tumor that Hospital (Jiangxi, China) were included in the present retro‑ originates in the bones (1,2). Each year in the United States spective 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 Correspondence to: Dr Zhi‑Wei Tao, Department of Bone and preoperative radiotherapy or chemotherapy were excluded. The Soft Tissue Oncology, Jiangxi Cancer Hospital, 591 Beijing East surgically removed tumor and adjacent normal tissue (5 mm Road, Nanchang, Jiangxi 330029, P.R. China distance from the tumor margin) was immediately fixed with E‑mail: [email protected] 4% formalin at room temperature for 48 h and the diagnosis of Abbreviations: CENPF, centromere protein F; IHC, osteosarcoma confirmed by two independent pathologists. The immunohistochemistry; RT‑qPCR, reverse transcription‑quantitative patients provided written consent for postoperative specimens PCR; shRNA, short hairpin RNA; OS, overall survival; DFS, to be used for scientific research before the operation. All disease‑free survival experiments were approved by The Human Ethics Committee of Jiangxi Cancer Hospital. Key words: CENPF, osteosarcoma, prognosis, biomarker Pathological evaluation. All clinical and clinicopathological characteristics were collected from the medical record of the 2 ZOU et al: CENPF IS LINKED TO AGGRESSIVE OSTEOSARCOMA patients and the staging of tumors were performed according ATT CAA GAG CTT GAA GGA CA‑3') and scrambled vector to The American Joint Committee on Cancer (11). The expres‑ (non‑ targeting sequence, 5'‑AGG TTA AGT CGC CCT CGC sion levels of CENPF in osteosarcoma were determined by TCG AG‑3') were assembled by Shanghai GenePharma Co., two independent pathologists. Ltd. The lentivirus vectors (C06002; Shanghai GenePharma Co., Ltd.; 3rd generation) were transfected into 293T cells Bioinformation analysis. For the mRNA expression levels using X‑tremeGENE HP DNA (cat. no. 6366236001; of CENPF in osteosarcoma, the boxplot and survival plots Roche Diagnostics). The supernatant was harvested 48 h were plotted by the Gene Expression Profiling Interactive post‑ transfection to concentrate lentivirus according to the Analysis (GEPIA) (http://gepia.cancer‑pku.cn/) database instructions. pMDL:VSVG:pRSV‑Rev: Sh‑vector were trans‑ using data from The Cancer Genome Atlas (TCGA) fected in a ratio of=5:3:2:5 in 100 mm Cell culture dishes, the database (https://www.cancer.gov/about‑nci/organization/ optical density (OD)260/280 of all plasmid =1.8‑1.9. ccg/research/structural‑genomics/tcga). The specific param‑ On the third day when MG‑63 and U‑2 OS were eters were as follows: Gene symbol=CENPF, |Log2FC| infected with lentivirus, MOI=1:10, 2 µg/ml of puromycin cut‑off=1, P‑value cut‑off=0.01, log scale=yes, jitter size=0.4 (cat. no. P8230; Beijing Solarbio Science & Technology Co., and matched normal data=match TCGA normal data. Ltd.) was added to screen positive cells for 1 week and the For Kaplan‑Meier survival plots, obtained from GEPIA drug concentration was maintained at all times. (http://gepia.cancer‑pku.cn/), the specific parameters were as follows: Methods=overall survival (OS) or disease‑free Reverse transcription‑quantitative (RT‑q)PCR. Total survival (DFS), group cut‑off=median, hazards ratio=yes, RNA was extracted from all cells, including wildtype and 95% confidence interval and axis units=months. transfected MG‑63 and U2‑OS cells using TRIzol® reagent (cat. no. 15596026; Invitrogen; Thermo Fisher Scientific, IHC analysis. The tumor tissues and adjacent tissues were Inc.) and cDNA was synthesized using First Strand cDNA cut into ~3‑mm thick sections and fixed in 4% formalin for Synthesis kit (cat. no. K1621; Thermo Fisher Scientific, Inc.) 24 h at room temperature. Subsequently, 4‑µm sections were according to the manufacturer's protocol. ABI 7900HT QPCR cut from the blocks, baked at 70˚C for 30 min, dewaxed with Cycle and FastStart Universal SYBR® Green Master (Rox) dimethylbenzene for 30 min and rehydrated with a decreasing (Roche Diagnostics). The following thermocycling conditions gradient series (100, 95, 85 and 75%) of ethanol for 30 min. To were used: 95˚C for 5 min; 40 cycles of 95˚C for 30 sec and antigen retrieve, slides were heated in the citrate buffer at 100˚C 60˚C for 1 min; and 60˚C for 3 min. GAPDH was used for (pH 6.0) for 20 min in a microwave oven. To avoid interfer‑ normalization and relative expression was calculated using ence with endogenous peroxidase, the slides were immersed in the 2‑∆∆Cq method (12). The primer sequences used were as 3% hydrogen peroxide for 5 min at room temperature. Tissues follows: GAPDH forward, 5'‑CAT CTC TGC CCC CTC TGC were blocked using 10% goat serum (cat. no. E510009; BBI Life TGA‑3', reverse, 5'‑GGA TGA CCT TGC CCA CAG CCT‑3'; Sciences Corp.) for 20 min at room temperature, then incubated and CENPF forward, 5'‑CTC TCC CGT CAA CAG CGT TC‑3', with anti‑CENPF antibody (1:200 dilution; cat. no. ab224813; reverse, 5'‑GTT GTG CAT ATT CTT GGC TTG C‑3' (13). Abcam) at 4˚C overnight. After washing with PBS three times, slides were incubated with horseradish peroxidase‑labeled Immunoblotting. All protein was obtained from cells, secondary antibody at room temperature for 30 min (1:1,000; including wildtype and transfected MG‑63 and U2‑OS cells goat anti‑rabbit IgG; cat. no. ab6721; Abcam). Thereafter, slides using RIPA buffer (cat. no. R0010; Beijing Solarbio Science were stained using 3,3‑diaminobenzidine reagent for 2 min & Technology Co., Ltd.) according to the manufacturer's at room temperature, followed by counterstaining with hema‑ protocol. Protein concentration was determined using the toxylin for 1 min at room temperature. Finally, all sections were Bradford Protein Assay kit (cat. no. PC0010; Beijing Solarbio sealed with neutral balata and images were captured under a Science & Technology Co., Ltd.). Total protein (30 µg/lane) light microscope. The H‑score system was used to evaluate the was separated by 10% SDS‑PAGE, transferred onto a PVDF IHC score. Patients were divided into high (H‑score ≥200) or membrane, and blocked with 5% fat‑free milk in TBST buffer low CENPF expression groups (H‑score <200). (TBS buffer with 0.5% Tween‑20) for 1 h at room temperature. Then, the membranes were incubated with anti‑CENPF anti‑ Cell lines. The human osteosarcoma cell lines MG‑63 body (1:1,000 dilution; cat. no. ab224813; Abcam) overnight and U‑2 OS were used in the present study.
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