아주대학교

목차

TABLE OF CONTENTS
ABSTRACT----------------------------------------------------------------------------------------------ⅰ
TABLE OF CONTENTS-----------------------------------------------------------------------------ⅲ
LIST OF FIGURES------------------------------------------------------------------------------------ⅴ
LIST OF TABLES-------------------------------------------------------------------------------------ⅶ
Ⅰ. INTRODUCTION----------------------------------------------------------------------------------1
A. Structure of Met receptor tyrosine kinase------------------------------------------------------------1
B. Aberrant activation of HGF-Met signaling in cancers---------------------------------------------3
C. Therapeutic strategies targeting Met in human cancers-------------------------------------------9
D. HGF-Met signaling in muscle differentiation and maintenance of rhabdomyosarcoma--11
E. Different splicing variants of Met---------------------------------------------------------------------13
F. D13Met, a novel alternative splicing variant of Met -----------------------------------------15
Ⅱ. MATERIALS AND METHODS-----------------------------------------------------------------17
A. Cell culture and transfections --------------------------------------------------------------------17
B. Reagents and antibodies--------------------------------------------------------------------------------18
C. RT-PCR----------------------------------------------------------------------------------------------------19
D. Immunoprecipitation and Western blotting--------------------------------------------------------19
E. Cell proliferation and thymidine incorporation assay--------------------------------------------20
F. Soft-agar colony formation assay----------------------------------------------------------------------21
G. In vivo tumorigenesis assay----------------------------------------------------------------------------21
H. Colony generation assay--------------------------------------------------------------------------------22
I. Migration assay--------------------------------------------------------------------------------------------22
J. Differentiation induction of primary cell or rhabdomyosarcoma cell lines------------------23
K. Lentiviral vector production------------------------------------------------------------------------------23
L. Statistical analysis------------------------------------------------------------------------------------------24
Ⅲ. RESULTS-------------------------------------------------------------------------------------------25
Part. 1. Inhibition of HGF-Met signaling by 13Met -------------------------------------------25
A. 13Met inhibits Met activation induced by HGF -----------------------------------------------25
B. 13Met inhibits HGF-Met signaling-dependent cell proliferation and colony
generation-----------------------------------------------------------------------------------------------------28
C. 13Met inhibits tumor growth in mice-------------------------------------------------------------32
D. Status of HGF-Met signaling is variable in rhabdomyosarcoma cells-----------------------34
E. Met silencing induces inhibition of ERK phosphorylation in RD cells-----------------------36
F. D13Met inhibits HGF-Met signaling in RH18 and RD cells------------------------------------38
G. Δ13Met reduces colony formation in RD cells----------------------------------------------------40
H. Δ13Met inhibits migration of RD cells-------------------------------------------------------------42
Part. 2. Induction of differentiation by D13Met ----------------------------------------------44
I. D13Met is induced during muscle differentiation-------------------------------------------------44
J. Abolishment of D13Met induction with specific siRNA results in delayed
differentiation of primary human skeletal muscle cells-------------------------------------------- 48
K. Overexpression of D13Met enhances differentiation in primary human skeletal muscle
cells---------------------------------------------------------------------------------------------------------51
L. Δ13Met is unable to induce differentiation of RD cells------------------------------------------54
M. Ectopic expression of D13Met hardly increases MHC expression in several Rhabdo-
myosarcoma cell lines under differentiation condition------------------------------------56
Ⅳ. DISCUSSION---------------------------------------------------------------------------------------58
Ⅴ. CONCLUSION-------------------------------------------------------------------------------------63
BIBLIOGRAPHY--------------------------------------------------------------------------------------64
국문요약-------------------------------------------------------------------------------------------------74