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ZWINT Is the Next Potential Target for Lung Cancer Therapy

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ZWINT Is the Next Potential Target for Lung Cancer Therapy Journal of Cancer Research and Clinical Oncology (2019) 145:661–673 https://doi.org/10.1007/s00432-018-2823-1 ORIGINAL ARTICLE – CANCER RESEARCH ZWINT is the next potential target for lung cancer therapy Fang Peng1 · Qiang Li2 · Shao‑Qing Niu1 · Guo‑Ping Shen1 · Ying Luo3 · Ming Chen4 · Yong Bao1 Received: 8 July 2018 / Accepted: 12 December 2018 / Published online: 14 January 2019 © Springer-Verlag GmbH Germany, part of Springer Nature 2019 Abstract Purpose We aimed to analyze the expression of ZWINT, NUSAP1, DLGAP5, and PRC1 in tumor tissues and adjacent tis- sues with public data. Methods The expression patterns of four genes were detected in cancer tissues and adjacent tissues by qRT-PCR. The overall survival analysis was used to explore these genes in lung adenocarcinoma and squamous cell carcinoma patients. Knockdown assays were used to select the most suitable gene among these four genes. Cell function assays with the knockdown gene were conducted in A549 and NCL H226 cells. The role of the knockdown gene in lung cancer was dissected in a mice tumor model. Transcriptome sequencing analyses with the knockdown gene were analyzed. Results Overexpression of these genes was significantly detected in cancer tissues (P < 0.01). Overall survival revealed that high expression of these genes is closely related with poor prognosis of lung adenocarcinoma patients (P < 0.05). Knock- down of ZWINT reduced proliferation in NCI H226 and A549 cells (P < 0.05). Knockdown also inhibited cell migration, invasion, apoptosis, and colony formation (P < 0.05). ZWINT knockdown reduced tumor volume (P < 0.05). Transcriptome sequencing of ZWINT knockdown-treated A549 and NCI H226 cells indicated that 100 and 426 differentially expressed genes were obtained, respectively. Gene ontology analysis suggested that binding, biological regulation, and multicellular organismal processes were the most enriched. KEGG analysis revealed that TNF, P53, and PI3K signal networks would be the most potential ZWINT-related pathways and were identified by Western blot analysis. Conclusions ZWINT may be a novel target for lung cancer therapy. Keywords Lung cancer · ZWINT · Differentially expressed genes · Gene ontology · KEGG Introduction Lung cancer is a serious threat to human life. This dis- ease has globally become the most common malignancy (McGuire 2016). About 1.6 million lung cancer patients are Fang Peng, Qiang Li, and Shao-Qing Niu contributed equally to this work. newly diagnosed worldwide each year. The mortality rate of lung cancer ranks first among malignant tumor-caused Electronic supplementary material The online version of this deaths (Sundar et al. 2014). The histological subtypes of article (https ://doi.org/10.1007/s0043 2-018-2823-1) contains supplementary material, which is available to authorized users. * Ming Chen 3 Department of Clinical Laboratory, Guangdong General [email protected] Hospital, Guangdong Academy of Medical Sciences, Guangzhou 510080, Guangdong, People’s Republic of China * Yong Bao [email protected] 4 Department of Radiation Oncology, Zhejiang Key Laboratory of Radiation Oncology, Zhejiang Cancer 1 Department of Radiation Oncology, The First Affiliated Hospital, 1 East Banshan Road, Hangzhou 310022, Zhejiang, Hospital of Sun Yat-sen University, 58 Zhongshan Road II, People’s Republic of China Guangzhou 510080, Guangdong, People’s Republic of China 2 Department of Organ Transplantation and General Surgery, Second Xiangya Hospital, Central South University, Changsha 410011, Hunan, People’s Republic of China Vol.:(0123456789)1 3 662 Journal of Cancer Research and Clinical Oncology (2019) 145:661–673 lung cancer are mainly divided into non-small cell lung et al. 2010), gastric cancer (Wu et al. 2017), breast cancer cancer (NSCLC) and small cell lung cancer (SCLC), (Chen et al. 2015a), and prostate cancer (Gordon et al. which accounts for about 85% and 15% of the lung can- 2017). Chen et al. suggested that NUSAP1 may be associ- cer patients, respectively. The 5-year survival rate of early ated with lung cancer occurrence and development (Chen NSCLC patients is about 40% after surgery (Ramalingam et al. 2015b). Although various genes are associated with et al. 2011). However, about 70% of patients have local or lung cancer, the detailed mechanisms of these four genes distant metastases at the time of diagnosis (Scagliotti et al. mentioned above have not been systematically studied. In 2008). Meanwhile, SCLC is more sensitive to radiotherapy this study, we aimed to describe the molecular functions and chemotherapy compared to NSCLC. To note, there are of four genes in the landscape of lung cancer. still limits for clinical treatments of SCLC. For example, SCLC is prone to drug resistance (Zarogoulidis et al. 2013). Therefore, it is necessary to perform more in-depth studies regarding clinical treatments of lung cancer. Method and materials Although the detailed mechanisms of lung cancer are still not fully understood, there are several genes that could Cells be potentially related with the occurrence and develop- ment of lung cancer. The Zw10 binding factor (Zwint MRC-5, H1975, NCI H226, and A549 cell lines were pur- White 10 interactor, Zwint) is a protein that regulates chased from ATCC (Virginia, USA). All the cells were cul- centromere division and is encoded by the Zwint gene. tured in RPMI 1640 media with 10% (v/v) FBS (Invitrogen, Zwint co-localizes with Zw10 on centromeres. Then, the Carlsbad, CA, USA). The cell lines were maintained in an complex attaches to the chromosome between the micro- incubator at 37 °C and 5% CO2. tubules of the spindle and spindle. Zwint is an important regulatory protein for chromosome movement and mitotic checkpoints. Previous studies have suggested Zwint Quantitative real‑time PCR (qRT‑PCR) over expression in breast and ovarian cancers (Woo Seo et al. 2015; Xu et al. 2016). PRC1 (Protein Regulator of Total RNA of MRC-5, H1975, NCI H226, and A549 cells Cytokinesis 1) is highly expressed during the S and G2/M were extracted with the MiniBEST Universal RNA Extrac- phases of mitosis and involved in cytokinesis. This is a tion Kit (Takara, Japan). The total RNA was immediately microtubule binding and bundling protein and essential to reverse transcribed to cDNA with the PrimeScript™ RT maintaining the mitotic spindle midzone (Hu et al. 2012). reagent Kit with gDNA Eraser (Perfect Real Time) (Takara, Genome-wide expression profile analysis suggests that Japan). The quality and quantity of total RNA were meas- PRC1 was significantly up-regulated in various cancers, ured using a NanoDrop 2000 (Thermo Fisher, USA). Sam- including cholangiocarcinoma, colon cancer, non-small ples were reextracted once the OD260/OD280 value was not cell lung cancer, pancreatic cancer, and breast cancer distributed between 1.8 and 2.1. qRT-PCR was carried out (Shimo et al. 2007). Moreover, PRC1 plays an important in a 50 µl reaction system, which included 25 µl 2xSsoAd- tumor-promoting role in the progression of many malig- vanced™ universal SYBR ® Green (BioRad, USA), 1 µl nant tumors, such as cervical cancer, prostate cancer, forward primer (10 µm), 1 µl reverse primer (10 µm), and hepatocellular carcinoma, and breast cancer (Kikuchi 50 ng cDNA. Thermal cycle conditions were as follows: et al. 2003; Lin et al. 2002; Nakamura et al. 2004; Obama 98 °C for 5 min; 40 cycles of 95 °C for 10 s, 60 °C for 30 s, et al. 2005). DLGAP5 is a microtubule-associated pro- and 72 °C for 20 s; and 12 °C forever. The primers used tein that is required for spindle formation during the cell in this study were: ZWINT (forward)—5ʹ-AAC TCC GGG cycle (Engeland 2018). DLGAP5 plays an important role AAG CCT TTG AG-3ʹ and ZWINT (reverse)—5ʹ-TTC TGG in the carcinogenesis of lung cancer cells (Shi et al. 2017). ACT GCT CTG CGT TT-3ʹ; NUSAP1 (forward)—5ʹ-CTT Overexpressed DLGAP5 may promote tumor radiotherapy GGGTCT GAA GGG GTC AC-3 ʹ and NUSAP1 (reverse)—5ʹ- resistance (Gomez et al. 2013). A recent study suggested TGCAGA CTT TCT GGC TGG AG-3 ʹ; DLGAP5 (forward)— that highly expressed DLGAP5 in NSCLC is associated 5ʹ-GGA ATC TGG GTG GCA AGT CA-3ʹ and DLGAP5 with patient prognosis (Schneider et al. 2017). In addition, (reverse)—5ʹ-TAG GCG ATT TCT CGC TGC AA-3ʹ; PRC1 the nucleolar and spindle-associated protein 1 (NUSAP1) (forward)—5ʹ-TGT CCA ATG CTA CGG CCA AT-3ʹ and is a microtubule and DNA-binding protein, which is PRC1 (reverse)—5ʹ-TTG CTG CCA GAA GGA GGA AG-3ʹ; involved in spindle assembly during mitosis. This protein and GAPDH (forward)—5ʹ-AAT GGG CAG CCG TTA GGA is also a key regulator to maintain normal cell cycle pro- AA-3ʹ and GAPDH (reverse)—5ʹ-GCGCCC AAT ACG ACC gression (Chou et al. 2011). Previous studies have shown AAA TC-3ʹ. The relative gene expression in different cell −ΔΔC that NUSAP1 is over-expressed in liver cancer (Satow lines was calculated via the 2 T method. 1 3 Journal of Cancer Research and Clinical Oncology (2019) 145:661–673 663 Knockdown treatments for 24 h. Subsequently, the migrated cells were fixed and incubated at 37 °C and 5% CO 2 for 1 h. Microscopy was Eight paired small hairpin RNAs against ZWINT, NUSAP1, used to analyze the number of migrated cells. DLGAP5, and PRC1 were designed and synthesized by Forevergen (Guangzhou, China). The following primers Cell apoptosis and colony formation were used in this study: siZWINT-1 (forward)—5ʹ-CCA GAG GAA ACG GAC ACA A-3ʹ and siZWINT-1 (reverse)— Annexin V-FITC Apoptosis Detection Kit (Sigma-Aldrich, 5ʹ-UUG UGU CCG UUU CCU CUG G-3ʹ; siNUSAP1-1 USA) was used to analyze cell apoptosis, following the man- (forward)—5ʹ-CCA AGA CUC CAG CCA GAA A-3ʹ and ufacturer’s instructions.
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