JBUON 2016; 21(4): 964-972 ISSN: 1107-0625, online ISSN: 2241-6293 • www.jbuon.com E-mail: [email protected] ORIGINAL ARTICLE Expression profiling of RE1-silencing transcription factor (REST), REST corepressor 1 (RCOR1), and Synapsin 1 (SYN1) genes in human gliomas Musteyde Yucebas1, Sunde Yilmaz Susluer1, Hasan Onur Caglar2, Tugce Balci1, Z. Ozlem Dogan Sigva1, Taner Akalin3, Nezih Oktar4, Tayfun Dalbasti4, Cigir Biray Avci1, Cumhur Gunduz1 1Ege University Medical Faculty, Department of Medical Biology, Bornova, Izmir; 2Ege University, Health Science Institute, Department of Stem Cell, Bornova, Izmir; 3Ege University Medical Faculty, Department of Pathology, Bornova, Izmir; 4Ege University, Medical Faculty, Department of Neurosurgery, Bornova, Izmir, Turkey Summary Purpose: The repressor element 1 (RE-1) silencing tran- Results: Means of relative expression for REST were as scription factor (REST) is a transcription factor which re- follows: 0.7898, 0.7606, and 0.7318 in DA, AO, and GBM presses the expression of neuronal differentiation-related groups, respectively. For RCOR1, expression means in DA, genes including SYN1 gene. CoREST, encoded by RCOR1 AO, and GBM groups were 0.7203, 0.7334, and 0.7230, re- gene, binds to the REST protein for remodeling of chroma- spectively. SYN1 expression means were as follows: 0.3936, tin structure. Although there is a relation among REST, 0.3192, and 0.3197 in DA, AO, and GBM groups, respective- RCOR1, and SYN1 genes, the role of these genes in glioma ly. Neither gain nor loss of copy numbers were detected for tumors is still unclear. In this study, expressions of REST, REST and RCOR1 genes in all groups. Copy loss for SYN1 RCOR1, and SYN1 genes were detected in primary cultures was detected in primary culture of a DA case. REST and derived from tumor samples of diffuse astrocytoma (DA), CoREST presented positive precipitation pattern on pro- anaplastic oligodendroglioma (AO), and glioblastoma mul- moter region of SYN1 gene. tiforme (GBM) cases. Conclusions: Expressions of REST and RCOR1 genes may Methods: Expression profiles were analysed by RT-qPCR downregulate SYN1 expression in gliomas. Low expression and the copy number variations were examined with qPCR pattern of SYN1 may maintain cancer stem-like phenotype in primary cultures. ChIP assay was performed to show which contributes to development of gliomas. binding characteristics of REST and CoREST proteins on promoter region of SYN1 gene. Key words: gene expression, gliomas, RCOR1, REST, SYN1 Introduction Gliomas are central nervous system tumors toma (grade IV) [1]. Classification system of glio- that originate from glial cells such as astrocytes or mas is based on the presence of necrosis in tumor oligodendrocytes which maintain and support the tissue, mitotic activity, and vascular or micro-vas- physiology of neuron cells. According to World cular proliferation [1]. Histopathological features Health Organization, gliomas are classified as as- of gliomas are associated with tumor recurrence, trocytoma (grade II), oligoastrcytoma (grade II), therapy response, and median survival of patients. oligodendroglioma (grade II), anaplastic astrocy- Glioblastoma tumors exhibit resistance to therapy toma/oligodendroglioma (grade III) and glioblas- and tumor recurrence after therapy. Besides, medi- Correspondence to: Sunde Yilmaz Susluer, PhD. Department of Medical Biology, Faculty of Medicine, Ege University, Izmir, Turkey, Tel: +90 232 390 2260, Fax: +90 232 342 05 42, E-mail: [email protected] Received: 26/12/2015; Accepted: 11/01/2016 REST, RCOR1 and SYN1 in gliomas 965 an survival for glioblastoma patients ranges from regulate the expression of the SYN1 gene. SYN1 14 to 16 months [2,3]. Studies indicate that gene gene expression is up-regulated during brain de- mutations, co-deletions, and extra-chromosomes velopment and this gene is expressed in adult are related with WHO grading and the prognosis neural tissue. Repression of the REST gene with of gliomas [4]. second-generation antisense oligonucleotides in- The repressor element 1 (RE-1) silencing tran- creases SYN1 expression in liver cells [21]. SYN1 scription factor (REST) is a transcription factor promoter has REST binding motifs and its expres- consisting of DNA binding and repressor domains sion is directly downregulated by REST protein [5,6]. DNA binding domain of REST binds to RE-1 [22]. Although there is a link among REST, RCOR1, sequence on promoter region of target gene, re- and SYN1 genes, interplay of these genes in glio- sulting in repression of expression [6]. It is known ma tumors is still unclear. that REST protein suppresses several neural dif- In this study, expressions of REST, RCOR1, ferentiation-related genes in the non-neuronal and SYN1 genes were analyzed in primary cell cells, while REST expression is down-regulated in cultures derived from tumor samples of DA, AO, neuronal precursor cells [5,7-11]. In carcinogen- and GBM cases. In order to demonstrate the cor- esis, REST can act as a tumor suppressor or an relation between copy number status and expres- oncogene. A splice variant of REST gene in small sion values, RT-qPCR was used. To show the bind- cell lung cancer cells (SCLC) causes production ing features of REST and CoREST transcription of truncated mature mRNA and this mRNA vari- factor on promoter region of SYN1 gene, chroma- ant down-regulates function of wild type REST tin immunoprecipitation assay was performed for protein [12]. Contrary to its tumor suppressor glioma derived primary cultures. function in SCLC, REST can act as an oncogene in neuroblastoma and medulloblastoma tumors and over-expression of REST has been identified Methods for these tumors [13,14]. Single nucleotide poly- Patients morphisms of REST leading to amino acid sub- stitutions are observed in primary and secondary Forty-nine patients with brain tumor (18 female, glioblastomas [15]. Furthermore, these polymor- 31 male) were recruited into the study. Informed con- sent was obtained from each patient and the study was phisms in REST are detected in other gliomas approved by the Local Ethics Committee of Ege Uni- such as astrocytoma, oligoastrocytoma, oligoden- versity, School of Medicine. The distribution of tumors drogliomas [15]. In addition to mutations, ampli- according to histopathology was as follows: 11 DA fication of REST is detected in glioblastomas [15]. (22.5%), 8 AO (16.3%), and 30 GBM (61.2%). The mean REST expression is found in GBM specimens and age was 53.00 ± 13.92 years. Demographic and clini- maintains self-renewal and tumor-initiating ca- cal features of cases are presented in Table 1. Prima- pacity of GBM stem-like cells. High REST expres- ry cell cultures of the tumor tissues were cultured in sion also induces aggressive properties of GBM BIOAMF-1 basal medium (Biological Industries, Israel) tumors in in vivo studies [16,17]. containing to 10,000U/ml penicillin, 10mg/ml strepto- Repressor domain of REST protein (RD2) in- mycin, 2 mM L-glutamine at 37oC in a humidified 95% teracts with REST corepressor 1 which is encoded air and 5% CO2 atmosphere. Expanded cell cultures of by the RCOR1 gene. REST and CoREST are assem- the third passage were used. bled with the histone demethylase LSD1/KDM1A and the histone deacetylases 1 and 2 (HDAC1/2) Isolation of total RNA and genomic DNA proteins to modify chromatin structure [6]. Chro- In order to detect mRNA expression of REST, matin repression by this complex leads to down- RCOR1, and SYN1 genes, total RNA were isolated from regulation of gene expression. For histone modi- primary tumor cell cultures. High Pure RNA Isolation fication, a complex consisting of both REST and Kit (Roche Applied Science, Mannheim, Germany) was CoREST may interact with different proteins such used to isolate total RNA from primary tumor cell cul- as SUMO and human positive coactivator 4 (PC4) tures according to manufacturer’s instructions. Total [18,19]. All three members of the CoREST family RNA was converted to cDNA with using Transcriptor proteins are expressed in brain tissue and this ex- First Strand cDNA Synthesis Kit (Roche Diagnostics, pression profile is observed at common territories Mannheim, Germany). cDNA samples were used for of brain. However, interactions of CoREST with gene expression analysis. For detection of copy number HDAC proteins in histone remodeling complex variations of REST, RCOR1, and SYN1 genes, genom- may show specific affinity depending on CoREST ic DNA samples were used. High Pure PCR Template family members [20]. REST and CoREST can also Preparation Kit for Genomic DNA (Roche Diagnostics, JBUON 2016; 21(4): 965 966 REST, RCOR1 and SYN1 in gliomas Table 1. Demographic and clinical features of glioma cases Gender Case number Age, years, mean Standard deviation Min age Max age (years) (years) F 18 49.78 15.40 15 73 M 31 54.87 12.87 30 79 Total 49 53.00 13.92 15 79 Diagnosis Grade Case number % Diffuse astrocytoma II 11 22.5 Anaplastic oligodendroglioma III 8 16.3 Glioblastoma IV 30 61.2 Total 49 100 F: female, M: male Mannheim, Germany) was used to obtain DNA samples pionChIP Antibody Kit (Sabiosciences, Germany-QIA- according to manufacturer’s instructions. GEN GmbH) was used as positive and negative control. Assays were performed for each antibody for 24 cases. Detection of gene expression and copy number variation Champion ChIP-One Day Kit (Sabiosciences, Germa- ny-QIAGEN GmbH) was used for ChIP assay. Exper- For the detection of gene expression, 2.6 µl cDNA imental steps were performed according to the man- were mixed with 7.4 µl Light Cycler PCR Master Mix ufacturer’s instructions. Primary tumor cell cultures and sterile bidistilled water was added to PCR mix. The obtained from tumor tissues were cultured until their expression studies of REST, RCOR1, and SYN1 genes numbers reached to 4-6x106. Chromatin structures in were performed with Real Time Online qRT-PCR ac- cell lysates were sheared with Sonicator S-4000 (Mis- cording to the Taqman protocol.