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Profiling Analysis Reveals the Crucial Role of the Endogenous Peptides

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Profiling Analysis Reveals the Crucial Role of the Endogenous Peptides OncoTargets and Therapy Dovepress open access to scientific and medical research Open Access Full Text Article ORIGINAL RESEARCH ProfilingAnalysis Reveals the Crucial Role of the Endogenous Peptides in Bladder Cancer Progression This article was published in the following Dove Press journal: OncoTargets and Therapy Weijian Li,1,* Yang Background: Peptide drugs provide promising regimes in bladder cancer. In order to Zhang,1,2,* Youjian Li,1,3,* identify potential bioactive peptides involved in bladder cancer, we performed the present Yuepeng Cao,4 Jun Zhou,1 study. Zhongxu Sun,1 Wanke Wu,5 Methods: Liquid chromatography/mass spectrometry assay was used to compare the endo­ Xiaofang Tan,5 Yang Shao,5 genous peptides between bladder cancer and normal control. The potential biological func­ tions of these dysregulated peptides are assessed by GO analysis and KEGG pathway Kaipeng Xie,5,6 Xiang Yan1,2 analysis of their precursors. The SMART and UniProt databases are used to identify the 1 Department of Nephrology and Urology, The sequences of the dysregulated peptides located in the functional domains. The Open Targets Children’s Hospital, Zhejiang University School of Medicine, National Clinical Research Center Platform database was used to investigate the precursors related to metabolic diseases. for Child Health, Hangzhou, People’s Republic Results: A total of 9 up-regulated peptides and 110 down-regulated peptides in bladder of China; 2Department of Urology, Drum Tower Hospital, Medical School of Nanjing cancer compared with normal control were identified (fold change > 1.2, P < 0.05). The MW University, Institute of Urology, Nanjing University, Nanjing, People’s Republic of China; of these dysregulated peptides ranged from 500 Da to 2500 Da and the MW of all identified 3Department of Urology Surgery, The People's peptides was below 3500 Da. The GO and KEGG pathway analysis indicated that these Hospital of Xuancheng City, Xuancheng, People's Republic of China; 4Department of dysregulated peptides could play an important role in bladder cancer. Our further analysis Critical Care Medicine, Jiangsu Cancer Hospital, revealed that 45HFNPRFNAHGDAN 57 derived from LGALS1 and those peptides derived Jiangsu Institute of Cancer Research, Nanjing Medical University AffiliatedCancer Hospital, from P4HB and SERPINA1 might be the promising diagnostic biomarkers and therapeutic Nanjing, People’s Republic of China; 5Nanjing targets of bladder cancer. Maternity and Child Health Care Institute, Women’s Hospital of Nanjing Medical Conclusion: In the present study, we have identified the profile of the peptides significantly University, Nanjing Maternity and Child Health dysregulated in bladder cancer. Moreover, using bioinformatic analysis, we found the pep­ Care Hospital, The AffiliatedObstetrics and Gynaecology Hospital of Nanjing Medical tides derived from LGALS1, P4HB and SERPINA1 could be the promising diagnostic University, Nanjing, People’s Republic of China; biomarkers and therapeutic targets of bladder cancer. 6Department of Women’s Health Care, The AffiliatedObstetrics and Gynaecology Hospital Keywords: peptide, therapeutic target, progression, bladder cancer of Nanjing Medical University, Nanjing Maternity and Child Health Care Hospital, Nanjing, People’s Republic of China *These authors contributed equally to this Introduction work Bladder cancer (BC) is the ninth most common cancer worldwide, including approxi­ 1,2 Correspondence: Xiang Yan mately 430,000 incident cases and 165,000 attributable deaths annually worldwide. Department of Nephrology and Urology, The Children’s Hospital, Zhejiang University It is a complex disease associated with high morbidity and mortality without effective School of Medicine, National Clinical Research therapy.3 Approximately 80% of BC are classified as non-muscle invasive BC Center for Child Health, Hangzhou, People’s Republic of China (NMIBC) depending on the degree of tumor infiltration in the vesical wall, while the Email [email protected] remainder are muscle invasive BC (MIBC).4 Patients with NMIBC have to undergo Kaipeng Xie invasive cystoscopy regularly after TURBT to detect tumor recurrence.5 Nevertheless, Nanjing Maternity and Child Health Care Institute, Women’s Hospital of Nanjing up to 70% of NMIBC patients still experience disease recurrence and progression.6 Medical University, Nanjing Maternity and Child Health Care Hospital, The Affiliated Furthermore, patients have to undergo radical cystectomy or chemotherapy in case of Obstetrics and Gynecology Hospital of Nanjing Medical University, Nanjing 210004, tumor recurrence and progression, which imposes a great deal of pain and injury on People’s Republic of China them.7,8 Therefore, in order to prevent BC progression more effectively, it is urgent to Tel/Fax +86-25-5222-6264 Email [email protected] reveal the potential mechanisms in the progression of BC. submit your manuscript | www.dovepress.com OncoTargets and Therapy 2020:13 12443–12455 12443 DovePress © 2020 Li et al. This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at https://www.dovepress.com/terms.php http://doi.org/10.2147/OTT.S281713 and incorporate the Creative Commons Attribution – Non Commercial (unported, v3.0) License (http://creativecommons.org/licenses/by-nc/3.0/). By accessing the work you hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For permission for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms (https://www.dovepress.com/terms.php). Li et al Dovepress Peptide is a type of organic compound produced by Materials and Methods endocrine glandular tissues or organs, containing 3–50 9 Patients and Tissue Specimens amino acids. As an important role, peptide participates The present study was conducted according to the recom­ in almost all physiological activities, including cell differ­ mendation of the Declaration of Helsinki and was approved entiation, immune regulation and, especially, tumor by the Ethics Committee of Nanjing Drum Tower Hospital. 10 formation. In recent years, the application of peptides Patients with a history of other cancers and any preoperative in the diagnosis and treatment of BC has aroused increas­ chemotherapy or radiotherapy were excluded. BC tissues and ing interest. Based on the different metabolomic character­ adjacent normal control tissues were collected from patients istics, two urinary peptides (AspAspGlyTrp and undergoing radical cystectomy. Finally, a total of 3 high- GlyCysAlaLys) were developed as early BC diagnostic grade BC tissues and the matched normal control tissues biomarkers.11 were collected (Table 1). All collected samples were placed Maria Frantzi et al exploits two urine-based peptide bio­ in frozen storage tubes and immediately snap-frozen in liquid marker panels for detecting primary and recurrent nitrogen and stored at −80°C until use. Each patient has urothelial BC.4 Some other endogenous peptides such as Six- signed the informed consent before we collected the samples. transmembrane epithelial antigen of the prostate (STEAP),12 13 antimicrobial peptide magainin II, DEP domain containing Peptides Extraction and Purification 14 1 (DEPDC1) and M-phase phosphoprotein 1 (MPHOSPH1) All collected samples were added to the protease inhibitor are promising peptides in the treatment of BC. mixture (Complete Mini EDTA-free, Roche, Basel, However, due to technical limitations, the mechanisms Switzerland) after isotonic PBS washing. The tissue specimen of peptides in the pathogenesis of BC have not been fully was cut into pieces and then ultrasonically homogenized for 20 studied in the past decades. With the technical development min. Then, we added 20% acetonitrile to the lysate to block the of proteomics, we have the opportunity to further investigate connection between the proteins and peptides. Then, the sam­ 15 the role of endogenous peptides in disease. Using pepti­ ples were centrifuged at 12,000 g in the condition of 4°C for 15 domics technology, a group of peptides have been identified min to obtain the supernatant. According to the manufacturer’s to function in cells.16,17 Therefore, as a new field focusing instructions, the samples were centrifuged by AMICON 10 on the function of endogenous peptides, peptidomics has kDa molecular weight cut off (MWCO) centrifuge filter emerged, which attracted increasing interests of researchers (Millipore). Then, the supernatant was filtered through recently.18 However, there is still very limited information a molecular filter, with the flow-through containing the pep­ on differentially expressed peptides between normal urothe­ tides. Afterward, the BCA method was used to measure the lium and BC tissue. Therefore, it is of great importance to concentration of the supernatant peptides. Lastly, supernatants perform an in-depth peptidomic analysis to reveal the were vacuum-dried by a Speed-vac system (RVC 2–18) and mechanism of BC progression. immediately frozen at −80°C for subsequent experiments. In the present study, we identified the endogenous pep­ tides that differentially expressed in BC. Furthermore, we TMT Labeling and LC-MS/MS Analysis analyzed the basic characteristic, cleavage sites patterns of The freeze-dried peptide was dissolved in 0.1% formic acid these dysregulated peptides. GO analysis, KEGG pathway and the solution was filtered through
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