Lamin subfamily differentially regulates the gene expression through the organization of 3D genome structure and splicing machinery

목차 (Table of Contents)

• 1. Introduction 1
• 1.1 Overview of Lamins 1
• 1.2 Lamin associated disease: Laminopathy 5
• 1.3 Lamin and chromatin organization 9
• 1.4 Lamins in transcription and DNA replication 12
• 1.5 Alternative splicing 14
• 1.6 Next Generation Sequencing 20
• 1.7 View of study 22
• 2. Materials and Methods 24
• 2.1 Oncomine database analysis 24
• 2.2 Kaplan-Meier analysis 24
• 2.3 Human tissue microarray and Immunohistochemistry (IHC) 24
• 2.4 Cell cultures 25
• 2.5 Western Blot 26
• 2.6 Immunostaining for FACS analysis 26
• 2.7 Immunocytochemistry 27
• 2.8 Generation of lamin knockout (KO) stable cell lines 28
• 2.9 Total RNA isolation and RT-qPCR 28
• 2.10 MTT assay and Cell migration assay 29
• 2.11 Live/dead cell assay 30
• 2.12 Annexin VFITC/propidium iodide (PI) apoptosis assay 30
• 2.13 Cell cycle analysis 31
• 2.14 RNA sequencing and bioinformatical analysis 31
• 2.15 Statistical analysis 32
• 2.16 Gene ontology and Gene Set Enrichment Analysis 32
• 2.17 Image analysis 33
• 3. Results 36
• 3.1 Up-regulation of lamin subfamilies in various lung cancers 36
• 3.2 Up-regulation of lamin families in lung cancer cells 41
• 3.3 Generation and validation of LMNA, LMNB1, and LMNB2 knockout cell lines 43
• 3.4 Mutual compensations between A- and B-type lamins in lamin deficient cells 45
• 3.5 Lamin deficit affects cell viability in lamin KO cells 48
• 3.6 Absence of lamin inhibits cell proliferation and migration ability 50
• 3.7 Lamin loss causes cell cycle arrest at G2/M contributing to growth inhibition in H1299 cells 52
• 3.8 Induction of apoptosis in lamin KO cells 54
• 3.9 Dynamic transcriptome profiling of lamin KO cells using RNA-seq 56
• 3.10 Extensive downregulation of lamin A tail domain binding partners 70
• 3.11 Remarkable increase of alternative splicing events in lamin KO H1299 cells 78
• 3.12 Exceptionally deterministic clustering of alternative splicing events at nuclear speckles 83
• 3.13 Lamin loss affects the granulation of nuclear speckles 86
• 4. Discussion 89
• 5. References 98
• 국문초록 109