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ZWINT: a Potential Therapeutic Biomarker in Patients with Glioblastoma Correlates with Cell Proliferation and Invasion

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ZWINT: a Potential Therapeutic Biomarker in Patients with Glioblastoma Correlates with Cell Proliferation and Invasion ONCOLOGY REPORTS 43: 1831-1844, 2020 ZWINT: A potential therapeutic biomarker in patients with glioblastoma correlates with cell proliferation and invasion LI YANG1,2, NA HAN1, XIAOXI ZHANG1, YANGMEI ZHOU1, RUI CHEN1 and MENGXIAN ZHANG1 1Department of Oncology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430030; 2Department of Oncology, Minda Hospital, Hubei Minzu University, Enshi, Hubei 445000, P.R. China Received September 9, 2019; Accepted February 26, 2020 DOI: 10.3892/or.2020.7573 Abstract. Glioblastoma (GBM) is the most aggressive primary effectively inhibited proliferation and invasion and induced intracranial tumor in adults. Chemoradiotherapy resistance apoptosis of GBM cells and notably suppressed GBM growth and recurrence after surgery are the main malignant progres- in vivo. Therefore, we speculate that ZWINT may be a poten- sion factors, leading to a high mortality rate. Therefore, the tial therapeutic biomarker for GBM, with NDC80 and PLK1 exploration of novel biomarkers and molecular mechanisms conjointly involved in regulating cell division and the mitotic of GBM is urgent. Differentially expressed genes (DEGs) of cell cycle. GBM were screened in a TCGA dataset. Homo sapiens ZW10 interacting kinetochore protein (ZWINT) was found to be Introduction upregulated in GBM, which was confirmed by immunohisto- chemical staining of a tissue microarray. Gene Ontology (GO) Glioblastoma (GBM) is the most common primary malignant annotation and Kyoto Encyclopedia of Genes and Genomes brain tumor in adults. High-grade GBM remains a devastating (KEGG) pathway enrichment analysis were performed using disease despite maximal therapy with surgery, radiation and the Database for Annotation, Visualization and Integrated chemotherapy with temozolomide (TMZ). GBM patients Discovery (DAVID) database. A protein-protein interaction present with only a 15‑ to 19‑month median overall survival (PPI) network was established by the STRING database, and rate due to chemoradiotherapy resistance and recurrence (1). hub genes were visualized by Cytoscape. The correlation Recent studies have identified several molecular alterations results were verified with the GSE15824 dataset. Bioinformatic involved in GBM carcinogenesis, progression, chemothera- analysis confirmed that ZWINT was significantly posi- peutic resistance and recurrence, such as IDH1, 1p/19q, MGMT, tively correlated with kinetochore protein NDC80 homolog ATRX and PTEN, but the precise mechanisms underlying (NDC80), serine/threonine-protein kinase PLK1 (PLK1) and this malignancy and its rapid recurrence have not been fully spindle and kinetochore associated complex subunit 1 (SKA1) elucidated (2-4). Targeting key genes and signaling pathways and together are involved in regulating mitosis and the cell is considered a promising approach for the diagnosis and treat- cycle of GBM. ZWINT expression was knocked down in ment of cancer. Therefore, finding potential biomarkers with U251 and U87 MG GBM cells by lentiviral vectors carrying a bioinformatic analysis contributes to a better understanding of small hairpin RNA (shRNA) targeting ZWINT. The effect of the occurrence and development of GBM. ZWINT silencing on cell proliferation, invasion and apoptosis Homo sapiens ZW10 interacting kinetochore protein was determined by the Celigo assay, MTT assay, Transwell (ZWINT) is a known component of the kinetochore complex assay, flow cytometry and caspase‑3/7 assay in vitro. A subcu- required for the mitotic spindle checkpoint and plays crucial taneous xenograft tumor model was established to explore roles in mitotic cycle maintenance (5). The kinetochore is the influence of ZWINT knockdown on GBM growth in vivo. a highly complex structure that is central to many essential Our preliminary study demonstrated that ZWINT knockdown activities during cell division. The kinetochore, a tri-laminar plate to which microtubules attach, connects chromosomes to the spindle to ensure the accurate segregation of chromosomes in mitosis and meiosis (6). ZWINT encodes a protein that is Correspondence to: Professor Mengxian Zhang, Department clearly involved in kinetochore function, possibly by regulating of Oncology, Tongji Hospital, Tongji Medical College, Huazhong the association between ZW10 and centromere complexes University of Science and Technology, 1095 Jiefang Road, Wuhan, during mitotic and mitotic prometaphase (7). It is known that Hubei 430030, P.R. China abnormal mitosis is a common feature of most malignan- E-mail: [email protected] cies. Although the exact role of the molecular makeup of the kinetochore and how individual components of the kineto- Key words: glioblastoma, bioinformatics, TCGA, GEO, ZWINT, chore interact with each other are unknown, growing evidence shRNA shows that ZWINT is often highly expressed in a number of human cancers and is linked with poor clinical prognosis and early recurrence (8-10). However, its role in human GBM 1832 YANG et al: ZWINT: A POTENTIAL THERAPEUTIC BIOMARKER IN GBM remains unclear. In our research, we aimed to investigate the The comparison criteria were as follows: Within -1<R<1, if expression of ZWINT and its biological significance in this R>0, indicated a positive correlation between the two genes; primary malignancy. R<0 indicated a negative correlation, and the greater the absolute value of R, the more significant was the negative Materials and methods correlation; and R=0 indicated nonlinear correlation. Dataset processing. TCGA (The Cancer Genome Atlas, Cell culture. The human GBM cell lines U251, U87 MG, https://cancergenome.nih.gov/) is a public repository for data A172, and U373 were obtained from the American Type storage that is freely available to users. A variety of human Culture Collection (ATCC). The specific origin of U87 MG cancer and tumor subtype genomic mutation profiles (11), tran- cells is unknown in the ATCC version. These four cell lines scriptomic data (12), and clinical data (13) have been generated, were authenticated using short tandem repeat (STR) profiling providing a systematic characterization of methylation (14), analysis following ISO 9001:2008 and ISO/IEC 17025:2005 miRNA expression (15), and oncogenic processes (16). Gene quality standards. The percent matches for U251, U373, A172 expression profiles of GBM were downloaded from the TCGA and U87 MG cells were 100, 89, 100 and 100%, respectively. dataset, which contains 529 GBM samples and 10 normal They were cultured in DMEM supplemented with 10% fetal samples. The data of the expression profile chip level 3 of bovine serum (FBS) (Ausbian, USA) and incubated at 37˚C in these samples were sorted out for analyzing the differen- a humidified atmosphere of 5% CO2. tially expressed genes (DEGs). However, multiple sets of data were assessed for certain samples in practice, thus the Immunohistochemistry microarray. A commercially available actual number of downloaded files was more than the original GBM tissue microarray (TMA) slide was purchased from samples (548 GBM samples vs. 10 normal samples). We used Shanghai GeneChem Co., Ltd., for IHC analysis. A specific P<0.05 and |FC| (fold change) ≥2 as the criteria, and the edgeR primary antibody against ZWINT was utilized for immu- (https://bioconductor.org/packages/release/bioc/html/edgeR. nohistochemistry (IHC) with a 2-step protocol. IHC scores html) (17,18) package in R 3.4.1 was used to identify DEGs were calculated according to the intensity and proportion of in the GBM samples compared with normal brain samples positive staining. Staining intensity was classified as 0 (nega- to finally obtain the DEG list. Another gene dataset, tive), 1 (weak), 2 (moderate) and 3 (strong). The positive cell GSE15824 (19), was downloaded from the NCBI GEO data- ratio of the stained cells was scored as 0 (<1%), 1 (1‑25%), 2 base (https://www.ncbi.nlm.nih.gov/geo/), and GPL570 was (26‑50%), 3 (51‑75%) and 4 (76‑100%). The multiplied result the platform file. Standardization data were carried out using of the two scores represented the protein levels of ZWINT, the RMA algorithm of the Affy (http://bioconductor.org/pack- and an immunoreactive score (IRS) >6 was defined as a high ages/release/bioc/html/Affy.html) (20) package in R software, expression level of ZWINT, while an IRS ≤6 indicated a low which were used for the subsequent analysis. level of ZWINT. GO and KEGG pathway analyses. Database for Annotation, Quantitative PCR (qPCR). Total RNA was extracted from cells Visualization and Integrated Discovery (DAVID) 6.8 using a TRIzol kit (cat. no. 3101-100, Pufei Biotechnology), (http://david.abcc.ncifcrf.gov/) is an online platform that and complementary DNA (cDNA) was synthesized with the is used for gene annotation, visualization and integrated M-MLV reagent (Promega). The SYBR Master Mix (Takara) discovery (21,22). Gene Ontology (GO) and Kyoto Encyclopedia and Mx3000P Real-Time PCR machine were used to perform of Genes and Genomes (KEGG) pathway enrichment analyses RT‑qPCR. The PCR primers (GeneChem) were ZWINT‑F, were implemented with the DAVID database. Using this 5 ' ‑ C A C GTA GAG GCC ATC AAA ATT GG‑3' and ZWINT‑R, comprehensive tool, we can understand the biological meaning 5 ' ‑ C G G AGT TGT GTC CGT TTC CT‑3'; GAPDH‑F, 5'‑TGA behind the DEGs more quickly and effectively. P<0.05 indi- CTT CAA CAG CGA CAC CCA‑3' and GAPDH‑R, 5'‑CAC cated a statistically significant difference. CCT GTT GCT GTA GCC AAA‑3'. Relative quantification was calculated using the ΔΔCq method
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