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Gene Silencing of USP1 by Lentivirus Effectively Inhibits Proliferation and Invasion of Human Osteosarcoma Cells

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Gene Silencing of USP1 by Lentivirus Effectively Inhibits Proliferation and Invasion of Human Osteosarcoma Cells INTERNATIONAL JOURNAL OF ONCOLOGY 49: 2549-2557, 2016 Gene silencing of USP1 by lentivirus effectively inhibits proliferation and invasion of human osteosarcoma cells JINBO LIU1, HONGJUN ZHU2, NING ZHONG3, ZIFENG JIANG4, LELE XU5, YOUPING DENG6, ZHENHUAN JIANG7, HONGWEI WANG8 and JINZHI WANG9 1Department of Orthopaedics, The Third Affiliated Hospital of Soochow University, Changzhou, Jiangsu 213003; 2Department of Thoracic Surgery, The First People's Hospital of Shangqiu, Shangqiu, Henan 476100; 3Department of Thoracic Surgery, Kunshan First People's Hospital Affiliated to Jiangsu University, Kunshan, Jiangsu 215000, P.R. China; 4Clinical Laboratories, The University of Chicago Medical Center, Chicago, IL 60637, USA; 5Department of Gastroenterology, The First Affiliated Hospital of Soochow University, Suzhou, Jiangsu 215200, P.R. China; 6Department of Medicine, Rush University Medical Center, Chicago, IL 60612, USA; 7Department of Orthopaedics, People's Hospital of Yixing City, Yixing, Jiangsu 214200, P.R. China; 8Department of Medicine, University of Chicago, Chicago, IL 60637, USA; 9Department of Cell Biology, School of Medicine, Soochow University, Suzhou, Jiangsu 215007, P.R. China Received August 17, 2016; Accepted October 20, 2016 DOI: 10.3892/ijo.2016.3752 Abstract. Osteosarcoma is the most frequent malignant bone and invasion in U2OS cells. Therefore, USP1 may provide a tumor, affecting the extremities of adolescents and young novel therapeutic target for the treatment of osteosarcoma. adults. Ubiquitin-specific protease 1 (USP1) plays a critical role in many cellular processes including proteasome degrada- Introduction tion, chromatin remodeling and cell cycle regulation. In the present study, we discovered that USP1 was overexpressed in Osteosarcoma is the most common bone malignancy in the 26 out of 30 osteosarcoma tissues compared to cartilage tumor pediatric age group, with a very high propensity for local inva- tissues and normal bone tissues. We then constructed a lenti- sion and distant metastasis (1,2). In recent years, despite the viral vector mediating RNA interference (RNAi) targeting development of new multimodal therapeutics, the prognosis USP1 and demonstrated that it significantly suppressed the for patients with this disease is generally poor (3,4). Therefore, mRNA and protein expression of the USP1 gene in U2OS effective therapeutic methods for osteosarcoma are urgently cells. Knockdown of USP1 inhibited the growth and colony- needed. forming, as well as significantly reduced the invasiveness of Ubiquitin (Ub), a kind of important signaling protein, U2OS cells. Western blot analysis indicated that suppression is involved in many cellular processes, including cell cycle of USP1 downregulated the expression of many proteins regulation, DNA damage response, chromatin remodeling and including SIK2, MMP-2, GSK-3β, Bcl-2, Stat3, cyclin E1, proteasome degradation. Similar to phosphorylation, ubiquitin Notch1, Wnt-1 and cyclin A1. Most of these proteins are asso- modification is reversible, and deubiquitinases or DUBs are ciated with tumor genesis and development. RNAi of SIK2 known to mediate ubiquitin removal (5). DUBs can remove significantly decreased SIK2 protein expression and inhibited Ub moieties from Ub-protein conjugates, resulting in reduced the ability of forming colonies, as well as induced apoptosis ubiquitination signaling (6). and reduced the invasiveness of U2OS cells. Collectively, our The misexpression of DUBs has been demonstrated to results suggest that silencing USP1 inhibits cell proliferation be associated with a number of human diseases, especially cancers (7). Among them, USP1 is one of the best-charac- terized human DUBs, which is involved in the control of cell differentiation and plays an important role in the regulation of Correspondence to: Dr Jinzhi Wang, Department of Cell Biology, the cellular response to DNA damage (8). School of Medicine, Soochow University, Suzhou, Jiangsu 215007, USP1 has been reported to be overexpressed in several P.R. China human malignant tumors (9,10). Moreover, USP1 has been E-mail: [email protected] identified as the DUB responsible for deubiquitinating Fanconi Dr Hongwei Wang, Department of Medicine, University of Chicago, anemia complementation group I (FANCI), Fanconi anemia Chicago, IL 60637, USA group D2 (FANCD2) and proliferating cell nuclear antigen E-mail: [email protected] (PCNA) in the DNA damage response. PCNA is monoubiq- uitinated in response to DNA damage that stalls progression Key words: osteosarcoma, USP1, RNA interference, SIK2 of the replication fork and then initiates recruitment of trans- lesion DNA synthesis (TLS) DNA polymerases in the DNA 2550 Liu et al: Silencing USP1 inhibits proliferation and invision of U2OS cells damage tolerance pathway (11,12). USP1 plays as a regulator sections were deparaffinized, rehydrated and immersed in 3% of PCNA ubiquitination in the DNA damage response. It may hydrogen peroxide solution for 15 min to block endogenous deubiquitinate the DNA replication processivity factor, PCNA, peroxidase activity. For antigen retrieval, the sections were thus, contributing to preventing unscheduled recruitment of boiled in EDTA buffer (pH 9.0) for 10 min. Histologic sections error-prone TLS DNA polymerases, as a safeguard against were pre-treated in 10% normal goat serum in TBS for 30 min error-prone TLS of DNA (13). at room temperature in order to block non-specific binding USP1-associated factor 1 (UAF1) stabilizes USP1 and allo- sites. Then sections were incubated with polyclonal antibody sterically increases its catalytic activity, which is very low in the against USP1 at a dilution of 1:100 in phosphate-buffered absence of the cofactor (14,15). In addition, UAF1 contributes to saline (PBS) at 4˚C for 1 h. After being rinsed five times with targeting USP1 to its nuclear substrates. USP1 performs its func- PBS, sections were incubated with goat anti-mouse IgG conju- tions in the context of a heterodimeric complex with UAF1 (16). gated with horseradish peroxidase at room temperature for RNAi is a powerful approach that silences the expression 1 h. Sections were developed with diaminobenzidene (DAB) of endogenous genes via distinct messenger RNA degrada- as a chromogen and counterstained with haematoxylin. The tion pathways. It may be a potential therapeutic agent for sections were analyzed under light microscopy. many kinds of diseases, including HIv, cerebrovascular and cardiovascular diseases, viral hepatitis, metabolic diseases, USP1 gene silence mediated by lentivirus-delivered shRNA. neurodegenerative disorders and cancers (17,18). The human USP1-specific small interfering RNA (siRNA) In the present study, specific-shRNA with lentivirus vector sequence is 5'-GAAAGCTCCACATCAATAA-3', designed was employed to knock down USP1 in osteosarcoma U2OS with an online Invitrogen software using the USP1 sequence cells, and the effects of USP1 silencing on cell growth and (GenBank code: NM_003600) as a reference. The non-silencing invasion were explored. sequence (5'-TTCTCCGAACGTGTCACGT-3') was used as a scrambled control. The recombinant lentiviral particles were Materials and methods regenerated in 293T cells by co-transfection of the modified pGCSIL-GFP viral vector together with the pHelper 1.0 and Main reagents. Mouse anti-human USP1 polyclonal antibody pHelper 2.0 plasmids using the Lipofectamine™ 2000 reagent. was purchased from the ProteinTech Group (Wuhan, China); The lentiviral particles were referred to as Lv-USP1 siRNA or mouse anti-human monoclonal antibody to Bcl-2 and mouse USP1-siRNA. We generated lentiviruses that express non- anti-human polyclonal antibodies to MMP-2, GSK-3β, Stat3, silencing-shRNA as controls and named them scramble siRNA cyclin E1, Notch1, Wnt-1 and cyclin A1 were purchased from or scr-siRNA. The osteosarcoma cells transfected with the ImmunoWay Biotechnology Co. (Plano, TX, USA); MG132 USP1-siRNA or scrambled siRNA were designated USP1- and mouse anti-human monoclonal antibody to USP1 were siRNA and scr-siRNA, respectively. For lentiviral transduction, obtained from Merck Millipore (Darmstadt, Germany); mouse U2OS cells were cultured in 6-well plates at 70% confluence anti-human monoclonal antibody to SIK2 were obtained and infected with control lentivirus or USP1-specific siRNA from BioLegend (San Diego, CA, USA); TRIzol reagent, lentivirus at MOI of 20. After 5 days of infection, U2OS cells Lipofectamine™ 2000, Opti-MEM and the SuperScript III expressing GFP protein were observed by fluorescence micros- reverse transcriptase (RT) kit were obtained from Invitrogen copy to determine the infection efficiency. Corp. (Carlsbad, CA, USA); Taq DNA polymerase was purchased from Fermentas, Inc. (Waltham, MA, USA); cell Sik2 siRNA preparation. The siRNA sequences were designed culture media and fetal bovine serum (FBS) were purchased by commercial software (Applied Biosystems/Ambion, Austin, from Gibco-BRL (Grand Island, NY, USA). TX, USA). For SIK2, the siRNA sense sequence is 5-CUA CCGAGAAGUACAAAUADTDT-3' and the antisense Tissue samples, cell lines and cell culture conditions. Tissue sequence is 5'-UAUUUGUACUUCUCGGUAGdTdT-3'. The samples from 30 osteosarcomas were obtained from patients siRNA sequences employed as negative controls were 5'-GG who underwent surgery at the Third Affiliated Hospital of CCUCAGCUGCGCGACGCdTdT-3' (sense) and 5'-GCGUC Soochow University. All participants who had undergone GCGCAGCUGGGCCAdTdT-3' (antisense). The selected surgical resection
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