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DNA Methylation and Gene Expression of COX7A1, SPINT1 and KRT81 in Glioblastoma and Brain Tissue

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DNA Methylation and Gene Expression of COX7A1, SPINT1 and KRT81 in Glioblastoma and Brain Tissue BIOLOGIJA. 2012. Vol. 58. No. 2. P. 71–78 © Lietuvos mokslų akademija, 2012 DNA methylation and gene expression of COX7A1, SPINT1 and KRT81 in glioblastoma and brain tissue Glioblastoma (GBM) is the most common primary brain tumor 1 Paulina Vaitkienė *, of the adult. Despite existing multimodal aggressive treatment 1 regiments the prognosis of patients with GBM remains very poor. Daina Skiriutė , Most patients die within two years after diagnosis. Changes in individual gene expressions frequently accompany malignant cell Wolf Mueller2 transformation. Therefore comparative gene expression analyses of tumorous and non-tumorous tissue may help identify genes of 1 Laboratory of Neurooncology and Genetics, Neuroscience Institute, significance in gliomagenesis. Epigenetic silencing of tumor sup- Lithuanian University pressor genes is one of important mechanisms of gene inactiva- of Health Sciences, tion during tumorigenesis. This study attempted to detect genes Eivenių st. 4, LT-50009 Kaunas, potentially regulated by promoter methylation in glioblastoma. Lithuania To this end we analyzed the expression of COX7A1, SPINT1 and KRT81 in glioblastomas and human brain tissue and investigated 2 Department of Neuropathology, their relation to promoter methylation of these genes. Institute of Pathology, We based candidate gene selection on bioinformatics, reverse Im Neuenheimer Feld 224, Heidelberg, 69120, Germany transcription-polymerase chain reaction (RT–PCR) and bisulfite sequencing. COX7A1, SPINT1 and KRT81 mRNA expression was analyzed in glioblastomas (n = 22) and human brain tissue (n = 2). COX7A1, SPINT1 and KRT81 promoter methylation was analyzed in selected glioblastoma samples, human brain and hu- man lymphocytes by bisulfite sequencing. We found a differential gene expression of COX7A1, SPINT1 and KRT81 in the glioblastoma samples. Normal fetal and adult brain featured a robust expression of these genes. Analysis of bi- sulfite-modified DNA of brain tissue confirmed that the SPINT1 promoter had only single scattered methylated CpG- dinucle- otides while the CpG- islands of the COX7A1 and KRT81 pro- moters were abundantly methylated in brain tissue despite robust gene expression. COX7A1 and KRT81 gene expression is independent of pro- moter methylation in glioblastoma. The data suggest the possi- bility of epigenetic regulation of SPINT1 in glioblastoma. Future work should focus on SPINT1 gene function in glial cells to sub- stantiate its potential tumor suppressor gene function in glio- mas. Key words: COX7A1, KRT81, SPINT1, glioblastoma, methyla- tion, expression * Corresponding author. E-mail: [email protected] 72 Paulina Vaitkienė, Daina Skiriutė, Wolf Mueller INTRODUCTION healthy tissue. To elucidate potential epigenetic gene regulation of COX7A1, SPINT1 and KRT81, Glioblastoma multiforme (GBM) is the most ag- we investigated their expression in human brain gressive form of malignant glioma. It is the most tissue and glioblastoma samples in relation to the common primary human brain tumor in the methylation status of their gene promoter. adult. Diffuse brain tissue infiltration and mo- lecular tumor cell properties of therapy resist- MATERIALS AND METHODS ance make treatment of these tumors especially difficult. Despite multimodal therapy with gross DNA and RNA isolation total resection in combination with adjuvant ra- 22 glioblastoma samples were obtained from dio- and chemotherapy prognosis of patients with Charité, Campus Virchow Klinikum from pa- GBM remains very poor. Median survival is about tients who underwent surgery in the Department one year following diagnosis. The analysis of gene of Neurosurgery and snap frozen. All GBM were expression and its regulation is one of the most histologically diagnosed according to the WHO intriguing fields in cancer research. Promoter criteria. Total RNA and DNA were isolated from methylation affects the expression of many genes snap frozen primary glioblastoma tissues using in normal and cancer tissue and may play an im- TRIzol (Invitrogen Life Technologies Inc) accord- portant role in gliomagenesis. Pharmacologic ing to the manufacturer’s recommendations. DNA manipulation of glioma cells with the demethylat- was extracted from snap frozen primary glioblas- ing agent 5’-aza-dC combined with genome wide toma tissues using Genomic DNA from Tissue expression profiling succeeded in unveiling novel Kit (Macherey-Nagel) according to the manufac- candidate genes, which are epigenetically regu- turer’s protocol. lated in cancer in general (Yamashita et al., 2002) and in gliomas in particular (Mueller et al., 2007; Reverse transcription–PCR analysis Elias et al., 2009). For cDNA synthesis 1 μg of total RNA was used COX7A1 (cytochrome c oxidase subunit VIIa for reverse transcription (random primer) using polypeptide 1), KRT81 (keratin 81) and SPINT1 SuperScriptTM First Strand Synthesis System (In- (serine peptidase inhibitor, Kunitz type 1) were vitrogen) according to the manufacturer’s instruc- among promising candidate genes identified in tions. The following primer sequences were used a previous wide study of such genome (Muel- (Table 1). ler et al., 2007). These genes revealed robust ex- All PCRs were performed in a 10-μl final vol- pression in normal brain samples, their gene pro- ume using AmpliTaq Gold PCR Master Mix (Ap- moter harbored a CpG-rich island and 5’-aza-dC plied Biosystems) with the following amplifica- demethylation was accompanied by a significant tion conditions: 95 °C for 5 min, and 36 cycles at up- regulation of their expression in glioma cells, 94 °C for 30 s, Tm for 40 s and at 70 °C for 30 s. suggesting epigenetic gene regulation (Muel- Amplified products were electrophoretically sep- ler et al., 2007). One important indication for a arated on 1.2% agarose gels, and visualized with potential role of a gene in the development of ethi dium bromide. cancer is its expression alteration compared to Table 1. cDNA primers Primer name Sequence Tm °C KRT81_cDNAf ATGCATCACCACCGTGTCGG 63 KRT81_cDNAr GCGCACCTTGTCGATGAAGG 63 COX7A1_cDNAf AGGACAAGGCAGAATGCAGG 63 COX7A1_cDNAr GGATGTCATTGTCCTCCTGG 63 SPINT1_cDNAf CATCAACTGCCTCTACGAGC 58 SPINT1_cDNAr ATCCCAGACCCTCCAAAGCC 58 GAPDH_cDNAf GGTTTTTCTAGACGGCAGGTCA 58 GAPDH_cDNAr TGGCAAATTCCATGGCACCGTCA 58 DNA methylation and gene expression of COX7A1, SPINT1 and KRT81 in glioblastoma and brain tissue 73 Table 2. Bisulfite modified DNA primers Primer name Sequence Tm °C KRT81_promoter_bisulfite GGTGTTTTGAGGAGTATATTGGAGT 53 modified_Frag_1f KRT81_promoter_bisulfite TAATAATACATAAAAAACTAAACCC 53 modified_Frag_1r KRT81_promoter_bisulfite GGGTTTAGTTTTTTATGTATTATTAT 55 modified_Frag_2f KRT81_promoter_bisulfite TATAATCCAAAACACCCACCTTATC 55 modified_Frag_2r Cox_promoter_bisulfite TTTGTAAAAATGTATTTTTTGGTAT 51 modified_Frag_1f Cox_promoter_bisulfite ACCTACATTCTACCTTATCCTCTTCC 51 modified_Frag_1r Cox_promoter_bisulfite GTGAGGTTTTTATGGGTTGGGT 53 modified_Frag_2f Cox_promoter_bisulfite AAAAAAATTTCCTAAATTAAAAAAAA 53 modified_Frag_2r SPINT1_promoter_Frag_1_methyl_f TTTTAGGTTTGGGGTTGGGAAAGTA 57 SPINT1_promoter_Frag_1_methyl_r CCTAATTTCTAATAAAACTAAATCAAC 57 Bisulfite modification formed on ten white colonies to validate the insert Sodium bisulfite modification was performed using primers: M13 Forward: GTAAAACGACG- using the CpGenome Universal DNA Modifi- GCCAG; M13 Reverse: CAGGAAACAGCTAT- cation Kit (Chemicon International) according GAC. ExoSAP-IT reagent (Affymetrix, USA) was to the manufacturer’s protocol. Universally me- used for PCR Product Cleanup. Preparation for thylated DNA (CpGenome Universal Methylated sequencing using SEQ Sequencing Reaction Kit DNA, Chemicon International) and unmethyla- (Montage, USA) according to the manufacturer’s ted DNA from normal blood samples were treated protocol. Fragments were sequenced and analysed with bisulfite by the same method. using Sequencher Software version 4.2. (Gene Codes Corporation, Ann Arbor, MI, USA). Bisulfite sequencing Initially, bisulfite-modified DNA of a target pro- RESULTS moter region was amplified. Primer sequences and conditions are shown below in Table 2. Reverse transcriptase polymerase chain reaction All PCRs were performed in a 20-μl final vo- (RT-PCR) is commonly applied to monitor gene lume using AmpliTaq Gold PCR Master Mix (Ap- expression. By RT-PCR gene expression can be in- plied Biosystems) with the following amplifica- vestigated with high accuracy and excellent repro- tion conditions: 95 °C for 5 min, and 36 cycles at ducibility. GAPDH (glyceraldehyde 3-phosphate 94 °C for 30 s, Tm for 40 s and at 70 °C for 2 min dehydrogenase) amplification was used as an in- and final extension 70 °C for 10 min. Amplified ternal control for cDNA quality and quantity. products were electrophoretically separated on Expression array data for COX7A1, KRT81 1.2% agarose gels, and visualized with ethidium and SPINT1 genes were verified by RT–PCR using bromide. cDNA isolated from 22 snap frozen glioblastoma The PCR product was plasmidincorporated samples. In addition, we investigated the expres- using One Shot Escherichia coli cells and the sion of these genes in cDNA isolated from normal TOPO TA Cloning Kit (Invitrogen Life Technolo- human brain. gies Inc). Cells were then plated and grown over- Data showed that the housekeeping gene night on prewarmed LB plates containing X-gal, GAPDH was equally expressed in all samples ampicillin and kanamycin. Colony PCR was per- confirming excellent cDNA quality and equal 74 Paulina Vaitkienė, Daina Skiriutė, Wolf Mueller Fig. 1. Differential expression
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