인간 줄기세포에서 microRNAs 발현 양상 및 기능 분석에 관한 연구

Abstract

Chapter 1 Stem cells are defined as self-renewing cell populations that can differentiate into multiple distinct cell types. However, hundreds of different human cell lines from embryonic, fetal and adult sources have been called stem cell; even through they range from pluripotent cells-typified by embryonic stem cells, which are capable of virtually unlimited proliferation and differentiation-to adult stem cell lines, which can generate a far more limited repertoire of differentiated cell types. MicroRNAs (miRNAs) are small non-coding RNAs that negatively regulate gene expression by complementary target mRNA transcripts. Multiple recent studies have found aberrant expression profiles of miRNAs in human stem cells. While a several target genes have been experimentally identified for some miRNAs in various stem cells. The global pattern of cellular functions and pathways affected by co-expressed miRNAs in human stem cells elusive. This study was to develop a computational approach to global analysis of the major biological processes, developmental processes, and signaling pathways that are most likely to be affected collectively by co-expressed miRNAs in human stem cells. We report results of computational analysis of four datasets of aberrantly expressed miRNAs in human embryonic stem cells (H9), adult stem cells (neural, mesenchymal, and CD34+ hematopoietic stem cells), embryonal carcinoma cells (NT2/D1), fetal normal lung fibroblast cells (Wi-38). Using the combinational target prediction algorithm miRGator and a two-step data reduction procedure we have determined Gene Ontology categories as well as biological function, developmental processes, and signaling genes. The group 1 is enriched location and developmental processes terms and the group 2 is enriched developmental processes, cell proliferation, and cell cycle terms. The group 3 is significantly enriched death and programmed cell death-related terms. Our global analysis of predicted miRNA targets suggests that co-expressed miRNAs collectively provide systemic compensatory response to the functional changes in stem cells by targeting a broad range of functional categories and signaling pathways known to be affected in cells. Such systems biology based approach provides new avenues for biological interpretation of miRNA profiling data and generation of experimentally testable hypotheses regarding collective regulatory functions of miRNA in stem cells. Chapter 2 Pluriotent stem cells, such as embryonic stem (ES) cells and embryonic carcinoma cells (NT2/D1), possess the remarkable property of differentiation potency, therefore this stem cell has great potential for investigating the stemness of stem cells. MicroRNAs (miRNAs) are recently discovered small noncoding RNAs with a broad spectrum of functions especially in development. Previously, we are reported novel miRNAs specifically expressed in human ES cells. Here we show that miR-302b indirectly regulates for Oct4, a pluripotent stem cell marker, and directly regulates for Cyclin D2, a developmental regulator during gastrulation and neurulation. Introduction of synthetic anti-miR-302b into undifferentiated human NT2/D1 cells leads to reduce of Oct4 expression and showed ahead of differentiation, even under the induced differentiation. During spontaneous and induced differentiation, Cyclin D2 protein but not mRNA expression is strongly increased, concurrent with the down-regulation of miR-302b and Oct4. These results suggested that miR-302b play an important role in maintaining of pluripotency and regulate the expression of cyclin D2 at post-transcriptional level in NT2/D1 cells. Respectively, these data are likely serves a similar purpose in human ES cells.; microRNA (miRNA)는 최근 발견된 small non-coding RNA로서 약 20~25개의 뉴클레오티드로 이루어져 있으며, 표적유전자의 3'-UTR (untranslated region)에 결합하여 posttranscription을 조절하는 것으로 알려져 있다. 현재까지 수백 개 이상의 miRNAs가 동물과 식물에서 발견되었으며 동물의 발달 및 분화과정에서 특이적으로 발현되고 있으나 아직 그 기능은 대부분 알려져 있지 않다. 줄기세포는 배아줄기세포와 성체줄기세포로 나뉘어질 수 있으며, 자가재생하는 세포로 전분화능을 가진 배아줄기세포를 포함한 여러 종류의 줄기세포에 대한 연구가 이뤄지고 있다. 본 연구에서는 인간 배아줄기세포 및 성체줄기세포에서 miRNA microarray를 통해 인간 배아줄기세포 특이적인 miR-302 및 miR-371 cluster와 성체세포 특이적인 let-7 family의 기능을 생물정보학적 방법을 통해 분석하였다. 인간배아줄기세포와 인가배아종양세포 특이적인 miR-302 cluster의 Gene Ontology 분석 결과 세포분화 및 증식에 관련된 유전자들이 선별되었다. 특히 miR-302 cluster 표적유전자 중 세포주기 조절 및 초기 장배형성 과정을 조절한다고 알려진 cyclin D2의 post-transcription을 조절하는 것을 확인하였다. 뿐만 아니라 miR-302b는 줄기세포의 줄기성 및 신경세포로의 분화를 조절하는 것으로 확인되었다. 이 결과를 통해 miR-302b는 cyclin D2의 post-transcription의 조절과 인간배아줄기세포와 배아종양세포의 분화능을 조절 할 것이다. 인간 배아줄기세포 및 성체줄기세포에서 miRNAs의 발현 양상 분석과 특이적으로 발현하는 miRNAs의 표적유전자의 분석을 통해 miRNAs의 기능을 유추할 수 있다. 그리고 이들 특이 miRNAs를 이용하여 세포의 운명을 조절이 가능할 것이다.