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Studies on the role of miRNAs in the TGF-β signaling pathway in lung cancer

  • 채동규 (대학원 생명과학과 분자생물학전공)

초록/요약

The transforming growth factor-β (TGF-β) signaling pathway plays an important role in carcinogenesis and various biological processes including cell proliferation, cell differentiation, and apoptosis. Therefore, TGF-β signaling dysregulation may contribute to pathologies such as cancer, cardiovascular disease and tissue fibrosis. Especially, SMADs protein and SMURF proteins play a key role in intracellular signaling of TGF-β signaling pathway. The regulatory mechanism of these genes influences TGF-β signaling activity. Thus, it is important to understand these regulatory mechanisms to treat a variety of diseases. miRNAs are small, non-coding RNA that function by regulating gene expression. As miRNAs may influence their target mRNA, they can participate in the regulation of biological processes and disease process. Recent studies have been reported that a number of miRNA are involved in the regulation of the members of TGF-β signaling pathway. The interaction between miRNA and TGF-β signaling influences the TGF-β pathway at multiple levels, such as members of signaling or TGF-β downstream genes. However, the regulatory mechanism of TGF-β signaling by miRNAs in largely unknown. In this study, I investigated the regulation of TGF-β signaling by miR-27a, miR-195 and miR-497 and the potential role of these miRNAs in lung cancer. SMAD2 and SMAD4 were direct targets of miR-27a, In addition, both miR-195 and miR-497 directly regulated SMURF2 gene. These results suggest that miR-27a which regulating SMAD2 and SMAD4 play important role as an oncogene. Downregulation of miR-27a significantly enhanced cell, colony and invasion. Whereas, miR-195 and miR-497 regulate SMURF2-dependent TβR1 ubiquitination and cause the activation of the TGF-β signaling pathway in lung cancer cells. Consequently, they suppress tumor growths in lung cancer. This study expands the regulatory mechanism of TGF-β signaling by miRNAs. My study will be useful in further cellular or clinical studies on miR-27a, miR-195 and miR-497 as a potential biomarkers for therapeutic targets and cancer diagnosis.

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목차

CONTENTS ⅰ
ABSTRACT ⅲ
LIST OF FIGURES ⅴ
LIST OF ABBREVIATION ⅶ
INTRODUCTION 1
1. TGF-β signaling pathway 1
1.1 The mechanism of TGF-β signaling pathway 1
1.2 SMAD protein 3
1.3 TGF-β signaling in cancer 4
2. MicroRNA 5
2.1 Biogenesis of miRNA 6
2.2 Regulation of TGF-β signaling by miRNAs
MATERIAL AND METHODS 9
1. Cell culture and transfection 9
2. Quantitative real-time PCR 9
3. Western blotting 11
4. Construction luciferase reporter plasmid and luciferase reporter assays 11
5. Ni-NTA Pulldown Assay 13
6. Flow cytometry 14
7. Cell proliferation assay 14
8. Isolation of Cell Surface Protein 15
9. Colony formation 15
10. Matrigel invasion assay 16
11. A549 Tumor Xenografts 16
12. Tissue immunofluorescence staining and TUNEL assay 17
13. Statistical analyses 17
RESULT
Part 1. MiR-27a regulates the TGF-β signaling pathway by targeting SMAD2 and SMAD4 in lung cancer 18
The alteration of miR-27a, SMAD2 and SMAD4 expression in lung cancer 19
MiR-27a directly modulate the SMAD2 and SMAD4 genes 23
MiR-27a promotes cell cycle by inhibition of TGF-β signaling 26
MiR-27a promotes cell proliferation and invasion 30
Part 2. MiR-195 and miR-497 suppress tumorigenesis and metastasis in lung cancer by inhibiting SMURF2-induced TGF-β receptor 1 ubiquitination 34
The expression of miR-195 and miR-497 in lung cancer patients 35
miR-195 and miR-497 regulate the SMURF2 gene by directly targeting the SMURF2 3′-UTR 38
miR-195 and miR-497 regulate the ubiquitination of TβR1 43
The activation of TGF-β signaling was regulated by miR-195 and miR-497 47
miR-195 and miR-497 regulate proliferation, colony formation and invasion 51
Overexpression of miR-195 and miR-497 suppresses xenograft tumor growth formation 55
Discussion 58
References 64
Abstract in Korean 74
Acknowledgements 76

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