Hassanudin Siti A, Ponnampalam Stephen N, Amini Muhammad N
Cancer Research Center, Institute for Medical Research, Jalan Pahang, 50588 Kuala Lumpur, Malaysia.
Oncol Lett. 2019 Feb;17(2):1675-1687. doi: 10.3892/ol.2018.9811. Epub 2018 Dec 7.
The aim of the present study was to determine the genetic aberrations and novel transcripts, particularly the fusion transcripts, involved in the pathogenesis of low-grade and anaplastic oligodendroglioma. In the present study, tissue samples were obtained from patients with oligodendroglioma and additionally from archived tissue samples from the Brain Tumor Tissue Bank of the Brain Tumor Foundation of Canada. Six samples were obtained, three of which were low-grade oligodendroglioma and the other three anaplastic oligodendroglioma. DNA and RNA were extracted from each tissue sample. The resulting genomic DNA was then hybridized using the Agilent CytoSure 4×180K oligonucleotide array. Human reference DNA and samples were labeled using Cy3 cytidine 5'-triphosphate (CTP) and Cy5 CTP, respectively, while human Cot-1 DNA was used to reduce non-specific binding. Microarray-based comparative genomic hybridization data was then analyzed for genetic aberrations using the Agilent Cytosure Interpret software v3.4.2. The total RNA isolated from each sample was mixed with oligo dT magnetic beads to enrich for poly(A) mRNA. cDNAs were then synthesized and subjected to end-repair, poly(A) addition and connected using sequencing adapters using the Illumina TruSeq RNA Sample Preparation kit. The fragments were then purified and selected as templates for polymerase chain reaction amplification. The final library was constructed with fragments between 350-450 base pairs and sequenced using deep transcriptome sequencing on an Illumina HiSeq 2500 sequencer. The array comparative genomic hybridization revealed numerous amplifications and deletions on several chromosomes in all samples. However, the most interesting result was from the next generation sequencing, where one anaplastic oligodendroglioma sample was demonstrated to have five novel fusion genes that may potentially serve a critical role in tumor pathogenesis and progression.
本研究的目的是确定与低级别和间变性少突胶质细胞瘤发病机制相关的基因畸变和新转录本，特别是融合转录本。在本研究中，组织样本取自少突胶质细胞瘤患者，另外还取自加拿大脑肿瘤基金会脑肿瘤组织库的存档组织样本。共获取了6个样本，其中3个为低级别少突胶质细胞瘤，另外3个为间变性少突胶质细胞瘤。从每个组织样本中提取DNA和RNA。然后使用安捷伦CytoSure 4×180K寡核苷酸阵列对所得基因组DNA进行杂交。人参考DNA和样本分别用Cy3胞苷5'-三磷酸（CTP）和Cy5 CTP标记，同时使用人Cot-1 DNA来减少非特异性结合。然后使用安捷伦Cytosure Interpret软件v3.4.2分析基于微阵列的比较基因组杂交数据中的基因畸变。从每个样本中分离的总RNA与寡聚dT磁珠混合以富集聚腺苷酸（poly(A)）mRNA。然后合成cDNA，并使用Illumina TruSeq RNA样本制备试剂盒进行末端修复、加poly(A)并连接测序接头。然后对片段进行纯化，并选择作为聚合酶链反应扩增的模板。最终文库由350 - 450个碱基对之间的片段构建而成，并在Illumina HiSeq 2500测序仪上使用深度转录组测序进行测序。阵列比较基因组杂交显示所有样本中几条染色体上有大量扩增和缺失。然而，最有趣的结果来自下一代测序，其中一个间变性少突胶质细胞瘤样本被证明有五个新的融合基因，这些基因可能在肿瘤发病机制和进展中起关键作用。
