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CUL4B Promotes Proliferation and Inhibits Apoptosis of Human Osteosarcoma Cells

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CUL4B Promotes Proliferation and Inhibits Apoptosis of Human Osteosarcoma Cells ONCOLOGY REPORTS 32: 2047-2053, 2014 CUL4B promotes proliferation and inhibits apoptosis of human osteosarcoma cells ZHI CHEN1*, BAO-LIANG SHEN2*, QING-GE FU3*, FEI WANG1, YI-XING TANG1, CANG-LONG HOU1 and LI CHEN1 1Department of Orthopedics, Changhai Hospital Affiliated to the Second Military Medical University, Shanghai 200433; 2Department of Orthopedics, Shanghai Jiading Central Hospital, Shanghai 201800; 3Department of Emergency and Trauma, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200120, P.R. China Received March 4, 2014; Accepted June 10, 2014 DOI: 10.3892/or.2014.3465 Abstract. Cullin 4B (CUL4B) is a component of the Introduction Cullin4B-Ring E3 ligase complex (CRL4B) that functions in proteolysis and is implicated in tumorigenesis. Here, we report Osteosarcoma is a primary bone malignancy with high rates that CUL4B is associated with tumorigenesis by promoting of metastasis, mortality and disability, and usually occurs in proliferation and inhibiting apoptosis of human osteosarcoma children and adolescents (1). The most common treatments cells. We performed RNA interference (RNAi) with a lentiviral for osteosarcoma are surgery, chemotherapy and biotherapy. vector system to silence the CUL4B gene using osteosarcoma However, the prognosis of osteosarcoma is still poor because SAOS-2 cells. The negative control included the normal of the high degree of malignancy, rapid disease progression target cells infected with the negative control virus whereas and early metastasis (2). the knockdown cells included the normal target cells trans- Gene amplification can be defined as increased copy fected with the RNAi target virus. We assessed the inhibition numbers of certain regions of the genome. Gene amplifica- resulting from the decreased expression of the CUL4B gene on tion often results in the upregulation of gene expression by the proliferation rate of SAOS-2 cells, and also evaluated the increasing the number of templates available for transcription. cell cycle distribution, apoptosis and clonability. Compared Gene amplification is commonly found in tumor cells and is with the negative control, the CUL4B gene expression was considered an important mechanism whereby tumor cells gain significantly inhibited in the SAOS-2 cells at the mRNA and increased levels of expression of critical genes. Thus, identi- protein levels in the knockdown group (P<0.01). Furthermore, fication and characterization of these genes should provide in the knockdown group, the cell proliferation rate and important insights into the pathobiology of cancer. clonability were also significantly inhibited (P<0.01). The Culllin 4B (CUL4B) is a component of the Cullin4B-Ring apoptosis rate increased significantly (P<0.05). A significant E3 ligase complex (CRL4B) that functions in proteolysis. It decrease in the number of cells in the G1 phase (P<0.01) and has been implicated in tumorigenesis since the core compo- significant increases in the S (P<0.01) and G2 phases (P<0.05) nents participate in a broad variety of physiologically and were observed. The silencing of CUL4B gene expression can developmentally controlled processes such as cell progres- effectively inhibit osteosarcoma cell proliferation and induce sion, replication and DNA damage response (3). In mammals, apoptosis. These findings may provide a novel biomarker for there are two Cullin 4 members, CUL4A and CUL4B, which the treatment of osteosarcoma. share 82% sequence identity. Although Cul4a-null mice do not exhibit evident developmental defects (4-6), CUL4A has been shown to target p53 and several cyclin-dependent kinase inhibitors, such as p21 and p27 for proteolysis in cultured cells (7-9) and was found to be highly expressed in breast and hepatocellular carcinomas (10,11). Loss-of-function mutation in the X-linked CUL4B, in contrast, causes mental retarda- Correspondence to: Dr Li Chen, Department of Hematology, tion, short state, absence of speech and other phenotypes in Changhai Hospital Affiliated to the Second Military Medical University, 168 Changhai Road, Shanghai 200433, P.R. China humans (12,13), and Cul4B knockout mice are embryonically E-mail: [email protected] lethal, indicating a unique function of CUL4B that cannot be compensated by CUL4A. Recently, Yang et al (14) showed that *Contributed equally depletion of CUL4B resulted in loss of not only H2AK119 mono-ubiquitination but also H3K9 tri-methylation and DNA Key words: CUL4B, osteosarcoma cells, proliferation, apoptosis methylation, leading to depression of a collection of genes, including the tumor-suppressor IGFBP3. Further experiments revealed that CUL4B promoted cell proliferation and invasion, 2048 CHEN et al: Function of CUL4B iN Osteosarcoma which are consistent with a tumorigenic phenotype, at least linearize the pGCSiL-GFP vector. We modified the double- partially by repressing IGFBP3. Further findings revealed stranded DNA after annealing and linked it with the that the expression of CUL4B was markedly upregulated in pGCSIL-GFP vector following the double digestion. We used samples of human cervical carcinoma and was negatively calcium chloride to prepare competent cells of Escherichia correlated with the expression of IGFBP3. coli afresh and cultured it at 37˚C for 16 h. We used computer- Although experiments have shown that CULB possesses an aided high-throughput cloning of bacteria in liquid medium intrinsic transcription repressive activity, (3,15) the exact role for sequencing (Shanghai Genechem Co., Ltd.). of CULB in the process and progression of osteosarcoma is still poorly understood. Here, we established an osteosarcoma Preparation and grouping of cells. The human osteosarcoma cell model in vitro, and transfected osteosarcoma SAOS-2 cell line SAOS-2 was cultured in the RPMi-1640 culture cells using CUL4B RNAi and assessed the apoptosis of solution with fetal calf serum (volume fraction 10%) and SAOS-2 cells. We demonstrated that CUL4B promotes cell incubated with 5% CO2 at 37˚C. The cells that remained in the proliferation and inhibits the apoptosis of osteosarcoma cells. logarithmic phase were divided into two groups: i) negative These results add to the understanding of the role of CUL4B control, in which the normal target cells were infected withthe in the proliferation and apoptosis of osteosarcoma cells and its negative control virus and ii) knockdown, in which the normal molecular mechanisms. target cells were infected with the RNAi target virus. Materials and methods Real-time PCR and western blotting to test knockdown effi- ciency. The human osteosarcoma SAOS-2 cells grew well on Cell line and culture conditions. Human osteosarcoma cell the day prior to viral introduction was recovered. The cell line SAOS-2 was purchased from the Shanghai Institute of the suspension was incubated with 5% CO2 at 37˚C. When the Chinese Academy of Sciences (Shanghai, China) and grown degree of cell fusion reached 30%, adequate viral load was in Dulbecco's modified eagle's medium (DMeM) with 10% introduced in the different groups, to a multiplicity of infec- (v/v) fetal calf serum, streptomycin (100 U/ml) and penicillin tion (MOi) of 100. Following incubation for three days, the (100 U/ml). DMeM, fetal bovine serum (FBS) and Dimethyl expression level of GFP was observed under a fluorescence sulfoxide (DMSO) were purchased from Gibco Biotechnology microscope. The culture was continued if the efficiency of (Gibco-BRL, MD, USA). Cultures were maintained at 37˚C in infection exceeded 50%. After incubation for five days, the an incubator with a humidified atmosphere of 5% CO2. cells were collected; the mRNA expression of the gene of interest was analyzed using real-time PCR for RNA interfer- Reverse transcription-polymerase chain reaction (RT-PCR) ence. The upstream primer sequence for the CUL4B gene was analysis of CUL4B gene expression in tumor cells. Total-RNA 5'-GGGAAAGGAATGGTGAA-3' while the downstream was extracted under RNase-free conditions (carried out primer sequence was 5'-TGCATAGAGCCGGTTAG-3'. The according to the operation manual for TRizol; invitrogen cell culture solution was aspirated and the cells were washed Corp., Carlsbad, CA, USA) with glyceraldehyde-3-phosphate twice in phosphate-buffered saline (PBS). An adequate amount dehydrogenase (GAPDH) as an internal reference. Primer of pre-cooled 2X lysis buffer was added. After deplating, the design was as follows: the CUL4B upstream sequence was cells were transferred to the tube and then lysed on ice for 5'-GGGAAAGGAATGGTGAA-3'; and the downstream 10-15 min. The protein concentration was determined and sequence was 5'-TGCATAGAGCCGGTTAG-3'. The GAPDH adjusted to 2 µg/µl. Next, 2X loading buffer was added to each upstream sequence was 5'-TGACTTCAACAGCGACAC sample and subsequently boiled for 5-10 min. Forty micro- CCA-3'; and its downstream sequence was 5'-CACCCTGTT grams of total protein was loaded into each well containing GCTGTAGCCAAA-3'. The Access RT-PCR kit was used to 10% SDS-polyacrylamide gel, subjected to electrophoresis at perform single-step reverse transcription and PCR amplifica- 30 mA for 2 min, and then transferred to a polyvinylidene tion. An aliquot of 5 µl of the amplified products was subjected fluoride (PVDF) membrane at 400 mA for 2 h. The membrane to electrophoresis on 2% agarose gel. The gels were examined was blocked with 5% milk in Tris-buffered saline (TBS) for under a UV lamp. 1 h at room temperature. The primary antibody was added and incubated with the membrane for 2 h at room temperature, and
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