KIF15 Is Involved in Development and Progression of Burkitt Lymphoma Zhao Wang1, Meiting Chen1, Xiaojie Fang1, Huangming Hong2, Yuyi Yao1 and He Huang1*
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Wang et al. Cancer Cell Int (2021) 21:261 https://doi.org/10.1186/s12935-021-01967-z Cancer Cell International PRIMARY RESEARCH Open Access KIF15 is involved in development and progression of Burkitt lymphoma Zhao Wang1, Meiting Chen1, Xiaojie Fang1, Huangming Hong2, Yuyi Yao1 and He Huang1* Abstract Background: Burkitt lymphoma (BL) is a highly aggressive, fast-growing B-cell non-Hodgkin’s lymphoma, manifested in several subtypes, including sporadic, endemic, and immunodefciency-related forms, the mechanism of which is still not clear. Abundant evidence reported that KIF15 was involved in the progression of human cancer. The emphasis of this study is to explore the functions of KIF15 in the development of BL. Methods: Firstly, tumor and normal tissues were collected for detecting expression of KIF15 in BL. Lentivirus-medi- ated shRNA knockdown of KIF15 was used to construct BL cell model, which was verifed by qRT-PCR and Western Blot. The cell proliferation was detected by CCK8 assay, cell apoptosis and cell cycle were measured through fow cytometry. Transwell assay was conducted to detect the migration. Results: We frst found that KIF15 is highly expressed in BL. Knockdown of KIF15 can inhibit proliferation and migra- tion, promote apoptosis and arrest the cell cycle. Moreover, KIF15 is involved in BL cell activity through regulating expression of apoptosis-related proteins (Caspase3, Caspase8, HTRA, IGFBP-6, p53, SMAC, sTNF-R1, TNF-β and Bcl-2) and downstream pathways, such as p-Akt, CCND1, CDK6 and PIK3CA. Conclusions: These fndings justify the search for small molecule inhibitors targeting KIF15 as a novel therapeutic strategy in BL. Keywords: KIF15, BL, Proliferation, Apoptosis, Migration Background that the three subtypes of BL (sporadic, endemic, and Lymphoma is a malignant tumor that originates in the immunodefciency-related) have diferent epidemiol- lymphopoietic system [1], which can be classifed into ogy, risk factors, and clinical manifestations [4]. Most non-Hodgkin’s lymphoma (NHL) and Hodgkin’s lym- BL exhibits considerable heterogeneity in clinical and phoma (HL) [2]. Depending on the origin of lymphocyte, pathological features [3, 4]. However, the treatment of it can be divided into B cell, T cell and NK cell lymphoma diferent types of BL is similar, and chemotherapy is the [2, 3]. Burkitt lymphoma (BL) is a highly aggressive B- main treatment method [5, 6]. In addition, the current NHL, which is characterized by the translocation and treatment regimen uses an intensive multi-drug treat- dysregulation of the MYC gene on chromosome 8 and ment regimen consisting of doxorubicin, an alkylating may involve multiple organ systems [4]. It is recognized agent, vincristine, and etoposide, whether short or long [5]. In addition to traditional therapies, a large number of *Correspondence: [email protected] molecular mechanisms have been gradually explored to 1 Department of Sun Yat-Sen University Cancer Center, State Key fnd efective targets. For example, Bouska et al., revealed Laboratory of Oncology in Southern China, and Collaborative that the BCR signaling pathway is a potential therapeu- Innovation Center of Cancer Medicine, 651 Dong feng East Road, Guangzhou 510060, Guangdong, China tic target in adult-BL [7]. Recently, Mrdenovic et al., pro- Full list of author information is available at the end of the article posed DZ-CIS to overcome CIS resistance in aggressive Zhao Wang and Meiting Chen are the co-frst author. B-cell BLs, and suggested that DZ-CIS could be used to © The Author(s) 2021. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Wang et al. Cancer Cell Int (2021) 21:261 Page 2 of 10 treat relapsed/refractory aggressive BL [8]. Although BL with DAB and hematoxylin for visualization. Images were is so far incurable, impressive response rates have been taken and analyzed under photomicroscope. achieved through molecular target drugs. Consequently, understanding the pathogenesis of BL is crucial for iden- Cell culture tifying potential therapeutic targets and formulating Te BL cell line Daudi and NAMALWA were obtained treatment protocols for BL. from the Cell Bank of the Chinese Academy of Sciences KIF15 belongs to the kinesin superfamily, a group of (Shanghai, China). Tey were cultured in RPMI1640 proteins that share highly conserved motor regions [9]. medium containing 20% fetal bovine serum (FBS) at the KIF15 (Kinesin-12) is a terminally oriented kinesin that atmosphere of 37 °C, 5% CO and 95% wet air. is localized in a mitotic manner to spindle microtubules 2 and chromosomes [10]. During mitosis, Kinesin is con- trolled spatially and temporally to ensure that it occurs Lentiviral shRNA vector construction and cell transfection precisely under steady inward and outward forces [11]. Tree RNA interference target sequences (shKIF-1: GCT However, overexpression of a few mitotic kinases may GAA GTG AAG AGG CTC AAA, shKIF-2: AGG CAG CTA result in further outward forces that contribute to a GAA TTG GAA TCA, shKIF-3: AAG CTC AGA AAG AGC series of adverse events, such as anaphase sister chro- CAT GTT) were designed with KIF15 as the template, matid separation, increased axial separation, and even- and the optimal kinetic parameter target was selected tual unipolar or bipolar or spindle formation [12]. Such for subsequent experiments. Oligo single stranded DNA events may lead to an unbalanced distribution of DNA, containing RNA interference target sequences was syn- aneuploidy, metastatic and invasive behavior, even cancer thesized and annealed to produce double stranded DNA. [12, 13]. Moreover, it has been demonstrated that KIF15 It is then directly connected to the digested lentivirus plays an important role in the development of several vector BR-V108 (bioscienceres Co. Ltd., Shanghai, China) types of human cancers such as pancreatic cancer [14], via its two restriction sites, Age I (NEB, Cat. # R3101L) lung adenocarcinoma [15] and breast cancer [16]. Never- and EcoR I (NEB, Cat. # R3101L). Te ligated products theless, limited data are available regarding KIF15 in BL, were transferred to the E. coli competent cells, and the especially in terms of its clinicopathological signifcance positive recombinant was identifed by PCR and sent to and its impact on molecular mechanisms. Herein, we sequencing for verifcation. Te correct positive recom- present our results eforts of KIF15 in BL and its clinical binant was expanded and cultured to obtain high purity relevance and mechanism. plasmid (EndoFree midi Plasmid Kit, TIANGEN, Cat. #DP118-2). Subsequently, 293 T cells were co-transfected Materials and methods with three plasmids (BR-V108, Helper 1.0 and Helper Immunohistochemical staining (IHC) 2.0) to obtain lentivirus. After 48 h the transfected len- tivirus was collected for concentration, purifcation and A total of 150 BL tissue chips were purchased from Xi quality testing, including physical status (color, viscosity), ’an Alina Biotechnology Co., Ltd. on April 1, 2019 from aseptic test and virus titer test. Te prepared lentivirus patients with difuse B-cell lymphoma. Te inclusion and was used to transfect the Daudi and NAMALWA cells. exclusion criteria of the sample were as follows: the inclu- Finally, the expression of green fuorescent protein was sion criteria were (1) clinical study of surgical treatment observed under a fuorescence microscope and the trans- of BL, regardless of language; (2) the age of the subjects fection efciency was evaluated transfection. was ≥ 18 years old; (3) patients with BL confrmed by his- topathology and/or cytology; (4) Tere was no contrain- dication of radiotherapy before treatment. Te exclusion Quantitative Real‑Time‑PCR (qRT‑PCR) criteria were (1) system analysis and review; (2) the age First, Daudi and NAMALWA cells were collected and of the subjects was less than 18 years old; (3) patients RNA was extracted by Trizol (Termo Fisher Scientifc who were not confrmed by histopathology and/or cytol- Cat. # 204211) according to the manufacturer’s instruc- ogy; (4) contraindications of radiotherapy. Te normal tions. Concentration and quality of extracted RNA were tissue samples used in this study are the tissues adjacent determined by Nanodrop 2000/2000C spectrophotom- to the tumor tissues of BL patients as control. Te tis- eter. Te cDNA was obtained by reverse transcription sue sections were deparafnized, repaired and blocked with the Promega M-MLV kit. Finally, qRT-PCR was per- with citric acid antigen, they were incubated with KIF15 formed using cDNA as the template and fusing curve was antibody at 4 °C overnight. After elution with PBS for 5 made. Te primer as follows, KIF15: 5′-CTC TCA CAG times, secondary antibody IgG (1: 400, Abcam, USA, # TTG AAT GTC CTTG-3′, 5′-CTC CTT GTC AGC AGA ab6721) was added and incubated at room temperature ATG AAG-3′, GAPDH: 5′-TGA CTT CAA CAG CGA CAC for 30 min. Tissue sections were subsequently stained CCA-3′, 5′-CAC CCT GTT GCT GTA GCC AAA-3′.