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. 2020 Jun;43(6):1831-1844.
doi: 10.3892/or.2020.7573. Epub 2020 Apr 2.

ZWINT: A potential therapeutic biomarker in patients with glioblastoma correlates with cell proliferation and invasion

Li Yang  1 , Na Han  1 , Xiaoxi Zhang  1 , Yangmei Zhou  1 , Rui Chen  1 , Mengxian Zhang  1
Affiliations

ZWINT: A potential therapeutic biomarker in patients with glioblastoma correlates with cell proliferation and invasion

Li Yang et al. Oncol Rep. 2020 Jun.

Abstract

Glioblastoma (GBM) is the most aggressive primary intracranial tumor in adults. Chemoradiotherapy resistance and recurrence after surgery are the main malignant progression factors, leading to a high mortality rate. Therefore, the exploration of novel biomarkers and molecular mechanisms of GBM is urgent. Differentially expressed genes (DEGs) of GBM were screened in a TCGA dataset. Homo sapiens ZW10 interacting kinetochore protein (ZWINT) was found to be upregulated in GBM, which was confirmed by immunohistochemical staining of a tissue microarray. Gene Ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed using the Database for Annotation, Visualization and Integrated Discovery (DAVID) database. A protein‑protein interaction (PPI) network was established by the STRING database, and hub genes were visualized by Cytoscape. The correlation results were verified with the GSE15824 dataset. Bioinformatic analysis confirmed that ZWINT was significantly positively correlated with kinetochore protein NDC80 homolog (NDC80), serine/threonine‑protein kinase PLK1 (PLK1) and spindle and kinetochore associated complex subunit 1 (SKA1) and together are involved in regulating mitosis and the cell cycle of GBM. ZWINT expression was knocked down in U251 and U87 MG GBM cells by lentiviral vectors carrying a small hairpin RNA (shRNA) targeting ZWINT. The effect of ZWINT silencing on cell proliferation, invasion and apoptosis was determined by the Celigo assay, MTT assay, Transwell assay, flow cytometry and caspase‑3/7 assay in vitro. A subcutaneous xenograft tumor model was established to explore the influence of ZWINT knockdown on GBM growth in vivo. Our preliminary study demonstrated that ZWINT knockdown effectively inhibited proliferation and invasion and induced apoptosis of GBM cells and notably suppressed GBM growth in vivo. Therefore, we speculate that ZWINT may be a potential therapeutic biomarker for GBM, with NDC80 and PLK1 conjointly involved in regulating cell division and the mitotic cell cycle.

Keywords: glioblastoma; bioinformatics; TCGA; GEO; ZWINT; shRNA.

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Figures

Figure 1.
Figure 1.
Hub genes and a significant module in the PPI network. (A) Hub genes. The ovals represent the DEGs, and the lines show the interactions between them. The red and yellow nodes represent the rank of hub genes from high to low according to the degree. (B) A significant module. The significant module contains DEGs that may play an important role in the PPI network. PPI, protein-protein interaction; DEG, differentially expressed genes.
Figure 1.
Figure 1.
Hub genes and a significant module in the PPI network. (A) Hub genes. The ovals represent the DEGs, and the lines show the interactions between them. The red and yellow nodes represent the rank of hub genes from high to low according to the degree. (B) A significant module. The significant module contains DEGs that may play an important role in the PPI network. PPI, protein-protein interaction; DEG, differentially expressed genes.
Figure 2.
Figure 2.
Correlation between the expression of ZWINT and the levels of other hub genes in GBM tissues. Correlation analysis of the public dataset GSE15824 showed that ZWINT mRNA expression was significantly positively correlated with the cell division and mitotic cell cycle markers NDC80, PLK1, NUF2, SKA1, SPC24, SPC25, SGOL1 and SGOL2. ZWINT, Homo sapiens ZW10 interacting kinetochore protein; GBM, glioblastoma; NDC80, NDC80 homolog; PLK1, serine/threonine-protein kinase PLK1; NUF2, NUF2 component of NDC80 kinetochore complex; SKA1, spindle and kinetochore associated complex subunit 1; SPC24, SPC24 component of NDC80 kinetochore complex; SPC25, SPC25 component of NDC80 kinetochore complex; SGOL1, shugoshin 1; SGOL2, shugoshin 2.
Figure 3.
Figure 3.
Expression level of ZWINT is upregulated in GBM tissues and cell lines. (A) The mRNA level of ZWINT was significantly higher in GBM samples than that noted in the normal samples based on the TCGA dataset (548 GBM samples vs. 10 normal samples. *P<0.05, t-test). (B) Representative immunohistochemistry (IHC) images of ZWINT expression in GBM tissue microarrays. Positive ZWINT staining was found in cytoplasmic tumor cells compared with normal brain tissues. (C) qPCR was used to determine ZWINT mRNA expression levels in glioma cell lines (ΔCt ≤12 indicates a significantly higher mRNA expression level of ZWINT in GBM cells). (D) Scoring results showed that ZWINT was overexpressed in GBM tissues, and the difference was statistically significant (**P<0.01). ZWINT, Homo sapiens ZW10 interacting kinetochore protein; GBM, glioblastoma.
Figure 4.
Figure 4.
ZWINT expression is effectively knocked down by shRNA. (A) Western blot bands reflect the efficiency of ZWINT silencing in 293T cells. (B) U251 and U87 MG cells were observed by light microscopy and fluorescence microscopy 3 days after infection. The green fluorescence represents infection efficiency, and over 80% of cells expressed GFP. (C) Confirmation of ZWINT knockdown in U251 and U87 MG cells by qPCR (**P<0.01). ZWINT, Homo sapiens ZW10 interacting kinetochore protein.
Figure 5.
Figure 5.
Effects of ZWINT knockdown on tumor cell proliferation. (A) Cell growth curves were plotted based on MTT absorbance. (B) Fluorescent photomicrographs were captured by measuring the cells with green fluorescence in each time series of cell growth, and then cell growth was graphed every day for 5 days by algorithms of the raw image data, as determined by a Celigo asssy (**P<0.01, shZWINT vs. shCtrl). ZWINT, Homo sapiens ZW10 interacting kinetochore protein.
Figure 5.
Figure 5.
Effects of ZWINT knockdown on tumor cell proliferation. (A) Cell growth curves were plotted based on MTT absorbance. (B) Fluorescent photomicrographs were captured by measuring the cells with green fluorescence in each time series of cell growth, and then cell growth was graphed every day for 5 days by algorithms of the raw image data, as determined by a Celigo asssy (**P<0.01, shZWINT vs. shCtrl). ZWINT, Homo sapiens ZW10 interacting kinetochore protein.
Figure 6.
Figure 6.
Effects of ZWINT knockdown on tumor cell invasion in U251 (A) and U87 MG (B) cells. Transwell assays showed that knockdown of ZWINT with shRNA (shZWINT) resulted in a significantly lower number of invading cells that migrated through the Matrigel-coated chambers compared to that of the vector control cells (shCtrl) (**P<0.01). ZWINT, Homo sapiens ZW10 interacting kinetochore protein.
Figure 7.
Figure 7.
Effects of ZWINT knockdown on tumor cell apoptosis in the U251 and U87 MG cell lines. (A) Cell death was measured by Annexin V-APC staining and flow cytometry. A higher percentage of apoptotic cells was noted in the shZWINT group vs. the shCtrl group. (B) Apoptotic cells were quantified by measuring caspase-3/7 activity (**P<0.01). ZWINT, Homo sapiens ZW10 interacting kinetochore protein.
Figure 7.
Figure 7.
Effects of ZWINT knockdown on tumor cell apoptosis in the U251 and U87 MG cell lines. (A) Cell death was measured by Annexin V-APC staining and flow cytometry. A higher percentage of apoptotic cells was noted in the shZWINT group vs. the shCtrl group. (B) Apoptotic cells were quantified by measuring caspase-3/7 activity (**P<0.01). ZWINT, Homo sapiens ZW10 interacting kinetochore protein.
Figure 8.
Figure 8.
Effects of ZWINT knockdown on tumor growth in vivo. (A) Images of the corresponding excised tumors. (B) Growth curve of tumor volumes and (C) histogram of tumor weights indicated that the shZWINT group tumors grew slower than the shCtrl group tumors (*P<0.05 and **P<0.01). ZWINT, Homo sapiens ZW10 interacting kinetochore protein.
Figure 8.
Figure 8.
Effects of ZWINT knockdown on tumor growth in vivo. (A) Images of the corresponding excised tumors. (B) Growth curve of tumor volumes and (C) histogram of tumor weights indicated that the shZWINT group tumors grew slower than the shCtrl group tumors (*P<0.05 and **P<0.01). ZWINT, Homo sapiens ZW10 interacting kinetochore protein.
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