Evaluation of CERS2 Gene as a Potential Biomarker for Bladder Cancer

Hindawi Disease Markers Volume 2019, Article ID 3875147, 8 pages https://doi.org/10.1155/2019/3875147 Research Article Evaluation of CERS2 Gene as a Potential Biomarker for Bladder Cancer Ahmed Faris Aldoghachi,1 Aminuddin Baharudin ,1 Umar Ahmad ,1 Soon Choy Chan,2 Teng Aik Ong,3 Rosna Yunus,4 Azad Hassan Razack,3 Khatijah Yusoff ,5,6 and Abhi Veerakumarasivam 1,6,7 1 Medical Genetics Laboratory, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Selangor Darul Ehsan, Malaysia Perdana University School of Foundation Studies, Perdana University, Selangor Darul Ehsan, Malaysia 3 Department of Surgery, Faculty of Medicine, University of Malaya, Wilayah Persekutuan, Kuala Lumpur, Malaysia 4 Histopathology Unit, Department of Pathology, Hospital Kuala Lumpur, Wilayah Persekutuan, Kuala Lumpur, Malaysia 5 Department of Microbiology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, Selangor Darul Ehsan, Malaysia 6 Malaysia Genome Institute, National Institute of Biotechnology Malaysia, Selangor Darul Ehsan, Malaysia 7 Department of Biological Sciences, School of Science and Technology, Sunway University, Selangor Darul Ehsan, Malaysia 2 Correspondence should be addressed to Abhi Veerakumarasivam; Received 16 November 2018; Revised 11 February 2019; Accepted 26 February 2019; Published 15 September 2019 Academic Editor: Tilman Todenhöfer Copyright © 2019 Ahmed Faris Aldoghachi et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The ceramide synthase 2 (CERS2) gene has been linked to tumour recurrence and invasion in many different types of cancers including bladder cancer. In this study, the expression levels of CERS2 in bladder cancer cell lines were analysed using qRT-PCR and the protein expression in clinical bladder cancer histopathological specimens were examined via immunohistochemistry. The potential utility of CERS2 as a predictive biomarker of response to oncolytic virotherapy was assessed by correlating the CERS2 mRNA expression to IC50 values of cells treated with the Newcastle disease virus (NDV), AF2240 strain. This study demonstrates that CERS2 is differentially expressed in different types of bladder cancer cell lines and that the siRNA-mediated downregulation of the expression of CERS2 reduces the migratory potential of UMUC1 bladder cancer cells. However, there were no significant correlations between the expression levels of the CERS2 protein with bladder cancer grade/stage or between the IC50 values of cells treated with NDV and CERS2 expression. Although the utility of CERS2 expression may be limited, its potential as an antimigration cancer therapeutic should be further examined. 1. Introduction Bladder cancer is ranked as the ninth most common cancer worldwide, and men are more than three times more likely to develop the disease as compared to women [1, 2]. Due to its high rate of recurrence and invasion, it has the highest cost of treatment per patient as compared to other malignancies [3]. Bladder cancer can be divided into two categories, nonmuscle-invasive bladder cancer and muscle-invasive bladder cancer. Nonmuscle-invasive bladder cancers account for 70% of all bladder cancers and are associated with low risk of progression and metastasis but high risk of recurrence. The treatment of nonmuscle invasive bladder cancer includes transurethral resection followed by a course of chemotherapy or immunotherapy to reduce the recurrence of the tumour [4, 5]. In contrast, muscle-invasive bladder cancers are associated with high rates of progression and metastasis and are usually treated by radical cystectomy if the tumour is organ-confined [4, 6]. To date, there are no prognostic or diagnostic biomarkers as well as predictive biomarkers of therapeutic response that are currently used in the mainstream management of bladder cancer patients [7, 8]. 2 The CERS2 gene, also known as the tumour metastasis suppressor gene 1 (TMSG1), contains Hox-like and TLC domains and is involved in the synthesis of long acyl chain ceramides. This gene was found to be expressed in 12 different types of human tissues, with the highest expression in the liver and kidney and the lowest expression in the colon, spleen, small intestines, thymus, and peripheral blood leukocytes [9]. Increased mRNA levels of the CERS2 gene were observed in metastatic cell lines as compared to nonmetastatic cell lines, implicating a role for CERS2 in metastasis [10]. Other studies have identified a correlation between CERS2 expression with the degree of recurrence and invasion in liver cancer [11], breast cancer [12], cervical cancer [13], pancreatic cancer [14], ovarian cancer [14], prostate cancer [15], and bladder cancer [11]. In a study conducted by Wang et al. [11], bladder cancer patients with low CERS2-expressing tumours were shown to have a poorer clinical prognosis. In recent years, the use of oncolytic viruses such as the Newcastle disease virus (NDV) has become an emerging cancer therapeutic modality. The idea of using NDV in the treatment of cancer has gained vast interest because these viruses are capable of selectively targeting and killing cancer cells [16, 17]. The NDV is a paramyxovirus, round in shape with a bilayer lipid envelope [18]. Oncolysis mediated by NDV occurs through either direct cancer cell killing via the formation of syncytium or indirect cancer cell killing by stimulating an antitumour immune response [19]. To date, no studies have been conducted to determine the potential of CERS2 as a biomarker that can predict NDV oncolytic response in bladder cancer. 2. Materials and Methods 2.1. Cell Culture of Bladder Cell Lines. The seventeen bladder cell lines used in this study were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and the European Collection of Authenticated Cell Cultures (ECACC, Salisbury, UK). Cells were cultured and maintained with growth media based on the suppliers’ recommendation. All the media were supplemented with fetal bovine serum (FBS) (Thermo Fisher Scientific Inc., Waltham, MA, USA). Cells were incubated at 37°C in a humidified atmosphere of 95% air and 5% CO2. 2.2. Analysis of CERS2 Expression in a Panel of Bladder Cancer Cell Lines. RNeasy Mini Kit (Qiagen, Hilden, Germany) was used for extracting the RNA of harvested cells following the manufacturer’s protocol. Quantification of the purity and concentration of the RNA was carried out using PCRmax Lambda spectrophotometer (PCRmax, Staffordshire UK). A total of 1 μg RNA was converted to cDNA using QuantiTect® Reverse Transcription Kit (Qiagen, Hilden, Germany). The cDNA synthesis was performed using a thermocycler (Eppendorf, Hamburg, Germany) with the following conditions: incubation for 15 and 3 minutes at 42°C and 95°C, respectively. The quantification of C (...truncated)