초병렬시퀀싱 방법을 이용한 인간 별아교세포의 microRNA 프로파일링 : MicroRNA Profiling of Human Primary Astrocytes using Massively Parallel Sequencing

다국어 초록 (Multilingual Abstract)

Background : Astrocytes have multifunctional properties including structural support for nervous tissue and maintaining metabolic environment. Most primary brain tumors such as astrocytomas and glioblastoma originate from astrocytes. MicroRNAs, a class of non-protein-coding RNAs that are ~22 nucleotides in length, have been implicated in the regulation of various biological processes including apoptosis, animal development, physiological functions such as insulin secretion, and differentiation. Recently, miRNAs have been implicated in cancer as important regulators of tumor suppressor genes and proto-oncogenes. The purpose of this study was to provide the fundamental data for molecular-biological understanding and development of novel diagnosis and therapies of diseases involved with astrocytes by profiling microRNAs of human primary astrocytes.

Methods : Human primary astrocytes were prepared from human fetal brains of 12-15 weeks gestation. Astrocytes were purified from other brain cells containing neurons, microglia and oligodendrocytes. Unlike hybridization-based methods, sequencing-based methods allow for direct discovery of novel microRNAs and detection of variations in mature microRNAs. In this study, massively parallel sequencing (MPS) was applied to microRNA profiling of primary human astrocytes.

Results : In total, genomic alignments from 32378 sequence reads were annotated as miRNA, piRNA, snRNA, snoRNA, scRNA, scaRNA, tRNA, rRNA, mRNA or unknown based on their genomic position. Among the 721 known human miRNAs registered in miRBase, 260 miRNAs were recovered in primary human astrocytes libraries. In addition, 27 novel miRNA candidates wered identified from unknown clusters of mapped sequence reads. The total set of novel miRNA candidates comprises 27 unique miRNA sequences from introns (12 sequences), intergenic regions (8 sequences), 5’ UTR of protein-coding genes (2 sequences), and 3’ UTR of protein-coding genes (5 sequence).

Conclusions : To the best of our knowledge, this presented work is the first to discribe a miRNA profiling　in human primary astrocytes using massively parallel sequencing. Our study might be fundamental for the comprehension of the molecular basis of the pathogenesis of such an aggressive tumor as glioblastoma, and give new clues to develop targeted therapies against this still untreatable cancer. Additional studies are now obviously needed to experimentally identify the targets of modulated miRNAs and to correlate them with brain tumor oncogenesis.

목차 (Table of Contents)

• Ⅰ. Introduction
• Ⅰ. 1. Types of cells in brain = 3
• Ⅰ. 2. Historical perspectives of neuroglia = 5
• Ⅰ. 3. Emerging roles of astrocytes = 7
• Ⅰ. 4. Transcriptomes = 9
• Ⅰ. 5. microRNA = 10
• Ⅰ. 6. Aims of the thesis = 12
• Ⅱ. Materials and Methods
• Ⅱ. 1. 세포배양 : Primary human astrocytes = 13
• Ⅱ. 2. RNA 분리 = 13
• Ⅱ. 3. small RNA library의 준비 = 14
• Ⅱ. 4. Massively parallel sequencing (MPS) = 17
• Ⅲ. Results = 18
• Ⅳ. Discussion = 27
• Ⅴ. Summary = 31
• Ⅵ. References = 32