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Original Article CDC5L Contributes to Malignant Cell Proliferation in Human Osteosarcoma Via Cell Cycle Regulation

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Original Article CDC5L Contributes to Malignant Cell Proliferation in Human Osteosarcoma Via Cell Cycle Regulation Int J Clin Exp Pathol 2016;9(10):10451-10457 www.ijcep.com /ISSN:1936-2625/IJCEP0031885 Original Article CDC5L contributes to malignant cell proliferation in human osteosarcoma via cell cycle regulation Yu Wang1,4*, Hong Chang2,4*, Di Gao3,4, Lei Wang4, Nan Jiang4, Bin Yu4 1Department of Orthopaedics, Chifeng Hospital, Inner Mongolia, China; 2Department of Orthopaedics, 421 Hospital of PLA, Guangzhou, China; 3Department of Orthopaedics, The University of Hong Kang, Shenzhen Hospital, Shenzhen, China; 4Department of Orthopaedics and Traumatology, Nanfang Hospital Southern Medical University, Guangzhou, China. *Equal contributors. Received May 9, 2016; Accepted July 22, 2016; Epub October 1, 2016; Published October 15, 2016 Abstract: Cell division cycle 5-like (CDC5L) has been reported in overexpressed in osteosarcoma (OS). However, its biological function in tumor biology was still unclear. Here, we firstly determined the expression of CDC5L in several OS cell lines, including Saos-2, SF-86, U2OS and SW1353, and found it was commonly upregulated in these four OS cells. Subsequently, Saos-2 and U2OS cells with higher CDC5L expression were transfected with interfering RNA tar- geting CDC5L. A set of functional assay was conducted on the two cell lines, including CCK-8, colony formation and flow cytometry assay. Our results indicated that CDC5L silencing significantly inhibited cell proliferation and arrested cell cycle at G2/M phase. Mechanically, Western blot analysis further confirmed knockdown of CDC5L remarkably down regulated the protein levels of CDK1, Cyclin B and PCNA. There findings further demonstrated that CDC5L play a crucial role in OS development and might be a promising therapeutic target of OS. Keywords: CDC5L, osteosarcoma, cell proliferation, cell cycle, CDK1, Cyclin B Introduction reported to shorten G2 phase of cell cycle, but its knockdown could delay mitotic entry by Osteosarcoma (OS) is the third most common arresting G2/M phase transition [6], as well as primary malignant bone tumor characterized as impair the pre-mRNA splicing efficiency. In addi- aggressive growth and proliferation [1]. For the tion, CDC5L has been demonstrated to regulate last few decades, despite remarkably advanc- expression and splicing efficiency of a set of es in the combination of surgery and chemo- genes involved in the mitosis and DNA damage therapy has been achieved, a significant pro- repair [7]. Furthermore, previous study sug- portion of OS patients shows high grade and gests that CDC5L is overexpressed in several poor prognosis [2]. Recently, molecular target- tumors, including glioma [8] and hepatocellular ed therapy has greatly advanced our knowl- carcinoma [9]. Moreover, knockdown of CDC5L edge of tumor pathogenesis and treatments, significantly inhibits tumor cell proliferation in glioma and hepatocellular carcinoma through which is particularly necessary to develop cell cycle arrest at G2/M phase. Recent studies effective therapeutic strategies for the treat- have described overexpressed CDC5L has a ment of OS. Therefore, it is urgent to identify poor prognosis in OS [10], but its exact biologi- novel molecular mechanisms associated with cal function and molecular mechanism remains OS initiates and progression. largely unknown in OS cells. As our best knowledge, uncontrolled tumor In the present study, we aimed to investigate growth is closely correlated with aberrant cell the biological function of CDC5L in human OS cycle control. Cell division cycle 5-like (CDC5L), cell lines and explore its molecular mechanism identified as a pre-mRNA splicing factor and by RNA interference technology. Our findings core component of human Prp19/Cdc5L com- might help us have a better understanding with plex, plays an important role in regulating cell the biological function of CDC5L and further division cycle [3-5]. Overexpression of CDC5L is confirmed CDC5L is an oncogene in OS. CDC5L contributes osteosarcoma Materials and methods Western blot analysis Cell cultures and transient transfection Whole proteins were obtained from cell sam- ples with 2X SDS lysis buffer containing 100 Human OS cell lines (Saos-2, U2OS, SW1353 mM Tris-Hcl (pH 6.8), 10 mM EDTA, 4% SDS and and SF-86) were provided by Cell Library, China 10% Glycine. The protein concentrations were Academy of Science and cultured in Dulbecco’s measured using Bicinchoninic acid (BCA) pro- modified Eagle’s medium (DMEM, Hyclone, tein assay kit (Thermo Fisher Scientific). App- USA) supplemented with 10% fetal bovine roximately 30 µg proteins were separated by serum. All cells were maintained in a 5% CO2 10% polyacrylamide-sodium dodecyl sulfate atmosphere at 37°C. (SDS-PAGE) electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes for The sequences of small interfering RNA (siRNA) 2.0 h at 300 mA. The membranes were blocked targeting human CDC5L (5’-GAGTGAAATTGC- with 5% non-fat milk in Tris-buffered saline for ACGTCAA-3’) and the negative control (NC) 1 h, and then incubated with primary antibod- siRNA were designed and synthesized by ies against CDC5L, CDK1, Cyclin B, PCNA and GeneChem (Shanghai, China). Saos-2 and 5 GAPDH overnight at 4°C. Then the membranes U2OS cells (1 × 10 cells per well) were seeded were washed three times for 10 min each time in six-well plates and then transfected with siR- with Tween 20-PBS and incubated with HRP- NAs using the Lipofectamine 2000 reagent conjugated secondary antibodies for 2 h at according to the manufacturer’s instructions room temperature. The expression levels of (Invitrogen, Carlsbad, CA, USA). The control OS proteins were analyzed using Super ECL De- cells were transfected with empty vectors. The tection reagent according to the manufacture’s cells were divided into three groups: The blank instruction. control (Con), empty vector (NC) and siRNA transfection (KD) groups. After culture for 48 h, Cell viability assay cells were collected and used for the following assays. To determine the effect of CDC5L on OS cell growth, Cell Counting Kit-8 (CCK-8) assay was Real-time PCR analysis performed on OS cells lines, Saos-2 and U2OS. Briefly, cells were seeded in 96-well plates at a Real-time PCR was used to validate the knock- density of 2000 cells per well and added 10 μL down efficiency induced by siRNA by determin- CCK-8 (Beyotime Biotechnology, China) after 1, ing CDC5L mRNA levels. Briefly, total RNA was 2, 3, 4 and 5 days, respectively. The optical extracted from cultured cells using TRIzol density (OD) of each well was determined using Reagent (Invitrogen) and used to synthesized a microplate reader at a wavelength of 450 nm. cDNA using MMLV reverse transcriptase (Pro- Each sample was repeated three times. mega, USA). Primers (forward: 5’-CAGGCTCCT- ACAGGACTACC-3’ and reverse: 5’-GGAACTTC- Colony formation assay CTGACCCTTGTG-3’) were designed to deter- mine CDC5L expression. GAPDH (forward: To evaluate the monolayer colony formation in 5’-TGACTTCAACAGCGACACCCA-3’ and reverse: OS cells, stably transfected cells from three 5’-CACCCTGTTGCTGTAGCCAAA-3’) was used as groups (Con, NC and KD) were seeded into six- endogenous control. Real-time PCR was per- well plates at density of 400 cells per well and formed on TAKARA TP800-Thermal Cycler cultured until the colonies were visible. Then Dice™ Real-Time System with 20 µl PCR reac- cells were fixed with 95% ethanol for 10 min tion mixture (10 µl 2 × SYBR premix ex taq, 0.5 and stained with 0.1% crystals purples. Cell µl primers (2.5 µM), 5 µl cDNA and 4.5 µl cDNA). colony formation was observed under a light The thermal cycling conditions were initially microscope. The number of colonies containing denatured at 95°C for 15 s, and 45 cycles of 5 more than 50 cells was manually counted. s at 95°C and 20 s at 60°C. Each reaction was Each sample was repeated three times. performed with triplicate samples in each group and analyzed individually relative to Flow cytometry analysis of cell cycle GAPDH. The relative expression of CDC5L was calculated using 2-ΔΔCt method [11]. Each sam- Flow cytometer (FACS Calibur, BD Biosciences) ple was repeated three times. combined with propidium iodide (PI) was used 10452 Int J Clin Exp Pathol 2016;9(10):10451-10457 CDC5L contributes osteosarcoma Figure 1. CDC5L was highly silenced in osteosarcoma cell lines. (A) The protein level of CDC5L in several osteosar- coma cell lines, including Saos-2, SF-86, U2OS and SW1353 using Western blot analysis (B and C) qPCR was used to determine the mRNA levels of CDC5L in Saos-2 and U2OS following CDC5L knockdown by siRNA transfection. (D and E) Western blot assay was used to determine the mRNA levels of CDC5L in Saos-2 and U2OS following CDC5L knockdown by siRNA transfection. Con: The blank control, NC: Empty vector and KD: siRNA transfection groups; all the results were represented as mean ± SD from three independent experiments. ***P < 0.001 as compared with NC cells; GAPDH was used as an internal control. to determine cell cycle progression in OS cells. Results Briefly, cells were reseeded on 6-cm dishes at a density of 2 × 105 cells per dish and fixed in CDC5L was significantly downregulated in OS 70% cold ethanol overnight at 4°C, followed by cells using siRNA transfection incubation with 300 µl PBS containing propidi- um iodide (PI) for 30 min at 37°C in dark, then To investigate whether CDC5L was correlated subjected to flow cytometry. Triplicate indepen- with OS progression, the proteins levels of dent experiments were performed. CDC5L were firstly determined in several OS cell lines using Western blot analysis. As shown Statistical analysis in Figure 1A, CDC5L was highly expressed in U2OS and Saos-2 than in other cell lines, which Quantitative data are analyzed using SPSS may be ascribed to specific cell type. Therefore, 13.0 software and presented as mean ± stan- U2OS and Saos-2 with higher CDC5L expres- dard deviations (SD) from at least three inde- sion were chosen to perform loss-of-function pendent experiments.
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