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Identification and in silico pathway analysis of the differentially expressed genes in bone marrow and adipose tissues of Cotl1 knockout mice

  • 김도완 (아주대학교 일반대학원)

초록/요약

Actin is the most important protein that consists of the actin cytoskeleton in living cells. The cytoskeleton plays a crucial role in cellular differentiation, proliferation, and growth, which are organized by actin dynamics, the process of assembly or disassembly of actin filaments regulated by numerous actin-binding proteins. Previous studies have implied the involvement of the association between actin cytoskeleton dynamics and cellular differentiation in the differentiation of mesenchymal stem cells (MSCs), which are multipluripotent stem cells that can differentiate into mesenchymal tissues, such as the bone and adipose tissue. Coactosin-like protein 1 (CLP or COTL1) is an actin-binding protein that binds strongly to actin filaments involved in actin dynamics, thereby regulating the cellular differentiation of MSCs. However, the genes possibly affected by Cotl1 expression and the associated pathways have not yet been elucidated. This study investigated the genomic association of Cotl1 with other genes involved in the cellular differentiation of MSCs through in silico pathway analysis by comparing bone marrow cells and adipose tissues of wild-type (WT) and Cotl1 knockout (Cotl1-/-) mice. RNA-sequencing analysis identified several differentially expressed genes upon Cotl1 deficiency, including 970 downregulated genes and 594 upregulated genes in bone marrow cells, as well as 251 downregulated genes and 1,427 upregulated genes in the adipose tissue. Analysis of diseases and biological functions via ingenuity pathway analysis revealed that cellular functions and skeletal system development were highly associated with Cotl1 deficiency in bone marrow cells, while cellular functions and metabolism of lipids were associated with Cotl1 deficiency in the adipose tissue. Furthermore, pathway analysis showed that Cotl1 deficiency affects various diseases and biological functions involved in the cellular development of MSCs in the bone marrow and adipose tissue. While formation of actin filaments, bone remodeling, adipogenesis, and differentiation of osteoclasts and adipocytes were predicted to decrease, the incidence of ossification, metabolic bone diseases, and disorders of lipid metabolism were predicted to increase upon regulation of a variety of genes associated with such diseases and biological functions. Additionally, twelve actin-, bone- and lipid-related core genes were identified and validated using quantitative reverse-transcription PCR. Furthermore, assessment of the phenotypes associated with bone and adipose tissue differentiation revealed that Cotl1-/- mice exhibited increased bone mineral density and reduced high-fat diet-induced weight gain compared to the WT mice. This study is the first to perform in silico pathway analysis of the bone marrow and adipose tissues in Cotl1-/- mice and suggest that Cotl1 may play an important role in bone and adipocyte differentiation, thereby highlighting that Cotl1 is a key gene that needs to be considered in the mechanistic study of ossification- and adipogenesis-related diseases.

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초록/요약

액틴(actin)은 세포골격(cytoskeleton)을 구성하는 가장 중요한 단백질로, 세포의 분화, 증식 및 성장에 매우 중요한 역할을 한다. 세포골격은 액틴 역동(actin dynamics) 이라고 불리는 액틴 필라멘트의 조립과 분해에 의해 만들어지며, 액틴 역동은 다수의 액틴 결합 단백질(actin-binding protein)에 의해 조절된다. 액틴 역동과 세포분화 사이의 연관성은 뼈와 지방 조직과 같은 중간엽 조직으로 분화할 수 있는 중간엽 줄기세포(mesenchymal stem cell: MSC)의 연구에서 잘 알려져 있다. Coactosin-like protein 1(COTL1)은 액틴 역동과 관련된 액틴 필라멘트에 결합하여 MSC 의 세포 분화를 조절하는 핵심적인 액틴 조절 단백질 중의 하나이다. 본 연구는, 야생형(wild type: WT)과 Cotl1 유전자 제거(Cotl1 gene knockout mouse; Cotl1-/-) 마우스의 골수 및 지방조직에서 발현에 변화를 나타내는 유전자를 발굴하고 이에 대한 in silico 경로 분석을 통해 MSC 의 세포 분화에 관여하는 COTL1 연관 인자를 찾는 것을 목적으로 하였다. 차세대염기서열분석장치(next-generation sequencer)를 이용한 RNA 염기서열 분석(RNA-sequencing)을 통해 전장(whole genome) 유전체의 RNA 발현 데이터를 얻은 후, WT 과 Cotl1-/- 마우스 조직 간의 차등 발현 유전자(differentially expressed gene: DEG)를 분석하였다. Cotl1-/- 마우스 골수에서 970 개 유전자가 WT 마우스에 비해 하향 조절(down-regulation)되었고, 594 개 유전자는 상향 조절(up-regulation)되었으며, 지방 조직에서는 251 개 유전자가 하향 조절되었고, 1,427 개 유전자가 상향 조절되어 있었다. IPA(Ingenuity Pathway Analysis) 방법을 활용하여 발굴된 DEG 에 대한 질병 및 생물학적 관련 기능 분석을 시행한 결과, Cotl1 유전자의 결핍에 의해 나타난 DEG 는 골수세포에서는 ‘세포 기능’ 과 ‘골격계 발달’과 연관성이 있는 유전자가, 지방 조직에서는 ‘세포 기능’ 과 ‘지질 대사’와 연관성이 있는 유전자가 대부분 포함되어 있음이 밝혀졌다. 또한, DEG 에 대한 in silico 경로 분석을 통해 Cotl1 결핍이 골수 및 지방조직에서 MSC 의 ‘세포 발달’과 관련된 다양한 질병 및 생물학적 기능에 영향을 미친다는 것을 확인하였다. 구체적으로, 액틴 필라멘트의 형성, 뼈 재구성(bone remodeling), 지방 형성, 파골세포 및 지방세포의 분화 기능이 감소할 것으로 예측되었으며, 골화(ossification), 대사성 골 질환 및 지질 대사 장애 등과 관련된 기능과 활성이 증가할 것으로 예측되었다. 이러한 결과의 종합적인 분석과 고찰을 통해 ‘액틴’, ‘뼈’, ‘지질’의 핵심어와 공통적으로 연관성이 높게 나타난 12 종의 유전자를 발굴하였고, 이들 유전자의 WT 마우스와 Cotl1-/- 마우스에서의 발현량의 차이를 정량 PCR(quantitative PCR: qRT-PCR) 실험을 통해 검증하였다. 상기의 결과를 바탕으로 Cotl1-/- 마우스의 뼈와 지방조직의 분화와 관련된 표현형(phenotype)을 조사한 결과, Cotl1-/- 마우스는 WT 마우스에 비해 골밀도가 증가되고, 고지방식이(high-fat diet)를 하였을 때 WT 마우스에 비해 체중증가가 억제되는 것을 발견하였다. 결론적으로, Cotl1 유전자 제거 마우스의 골수 및 지방 조직에서의 차등 발현 유전자 분석과 이에 대한 in silico 경로 분석을 통해, 액틴 결합 단백질인 COTL1 이 뼈와 지방세포의 분화에 중요한 역할을 한다는 사실을 밝혔다.

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목차

I. INTRODUCTION 1
II. MATERIALS AND METHODS 8
A. Animals 8
B. RNA isolation and next-generation RNA-sequencing 8
C. Gene mapping and identification of differentially expressed genes 9
D. Disease and biological function analysis 10
E. Pathway analysis 10
F. RNA isolation and quantitative reverse-transcription PCR 12
G. Examination of mouse bone and adipose tissue phenotypes 13
III. RESULTS 15
A. Identification of DEG profiles 15
B. Disease and biological function analyses of the bone marrow and adipose tissues 17
C. Pathway analysis of actin-related biological functions in the bone marrow and adipose tissues of WT and Cotl1-/- mice 19
C-1. Formation of actin filaments 19
C-2. Organization of actin cytoskeleton 22
C-3. Common gene network analysis of actin-related biological functions between the bone marrow and adipose tissue DEG sets 25
D. Pathway analysis of bone-related diseases and biological functions of the bone marrow and adipose tissues in WT and Cotl1-/- mice 27
D-1. Remodeling of bone 27
D-2. Differentiation of osteoblasts and osteoclasts 30
D-3. Ossification 34
D-4. Metabolic bone disease 37
D-5. Common gene network analysis of bone-related diseases and biological functions between the bone marrow and adipose tissue DEG sets 40
E. Pathway analysis of lipid-related diseases and biological functions in the adipose tissues and bone marrow cells of the WT and Cotl1-/- mice 42
E-1. Adipogenesis 42
E-2. Differentiation of adipocytes 45
E-3. Metabolism of lipid 48
E-4. Disorder of lipid metabolism 51
E-5. Common gene network analysis of lipid-related diseases and biological functions between the adipose tissue and bone marrow DEG sets 54
F. Core-gene network analysis of actin-, bone-, and lipid-related diseases and biological functions in the bone marrow and adipose tissues in WT and Cotl1-/- mice 56
G. Evaluation of the mRNA expression levels of core genes using in silico pathway analysis 58
H. Bone and adipose tissue phenotypes of mice 61
IV. DISCUSSION 63
V. CONCLUSION 72
REFERENCES 73
국문요약 88

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