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22대 대한민국 국회 국회정보길라잡이 국회의원검색
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Title Page

Contents

Abstract 12

Ⅰ. INTRODUCTION 14

1. Epithelial splicing regulatory protein (ESRP1) 14

2. Leucine-rich Repeat in Flightless Interaction Protein 2 (LRRFIP2) 18

3. Gastric cancer 21

4. The stream of the study 22

Ⅱ. MATERIALS AND METHODS 23

1. Cell culture and transfection 23

2. Plasmids 24

3. Generation of stable cell lines 25

4. RNA extraction, RT-PCR and real-time qRT-PCR 26

5. RNA-seq data analysis of gastric cancer cell lines and tissues. 27

6. RNA sequencing of LRRFIP2 (Δexon7) cell lines 29

7. GO, KEGG pathway, and PPI network analysis 31

8. Analysis of patient survival 32

9. Immunoprecipitation and immunoblot analysis 33

10. Crosslinking for protein interaction analysis 34

11. Cell migration and invasion assays 35

12. Quantification and statistical analyses. 36

Ⅲ. RESULTS 37

PART Ⅰ. Alternative Splicing Events Regulated by ESRP1 in Gastric Cancer 37

1. Expression of ESRP1 is associated with variant change of LRRFIP2 in gastric cancer cell lines 37

2. The relative frequencies of LRRFIP2 splicing are significantly associated with the expression levels of ESRP1 in gastric cancer patient tissues. 46

PART Ⅱ. The effect of LRRFIP2 variant change on tumor formation and metastasis 55

1. Overexpression of LRRFIP2 variant 3 induces liver metastasis, but not tumor growth. 55

2. Deletion of exon 7 in LRRFIP2 decreases the metastatic potential of gastric cancer cells. 62

PART Ⅲ. The regulatory mechanism of LRRFIP2 variant change on metastasis 65

1. Exon7 knockout in LRRFIP2 changes the transcriptome of gene expression patterns. 65

2. CARM1 is the LRRFIP2 variant 2-specific binding partner. 73

3. CARM1 interacts with ACTR in the transcriptional regulation of SERPINE1 in the presence of LRRFIP2 variant 3. 86

Ⅳ. DISCUSSION 91

Ⅴ. CONCLUSION 97

REFERENCES 98

ABSTRACT IN KOREAN 111

List of Figures

Fig. 1. ESRPs regulate markers and regulators of EMT. 15

Fig. 2. Roles of LRRFIP1/GCF2 in transcriptional repression, signal transductions, and regulation on the cytoskeletal system. 20

Fig. 3. High expressions of ESRP1 is correlated with short overall survival times of gastric cancer patients. 38

Fig. 4. Expression of ESRP1 and alternative splicing isoforms in18 gastric cancer cell lines. 40

Fig. 5. The splicing variants of LRRFIP2 in ESRP1-low and -high conditions. 42

Fig. 6. LRRFIP2 variants 2 and 3 are highly expressed in the 18 gastric cell lines and their expression levels are significantly correlated with... 43

Fig. 7. Expression levels of the two isoforms of LRRFIP2 in 18 gastric cancer cell lines. 45

Fig. 8. The expression levels of splicing variants are significantly associated with the expression levels of ESRP1 in gastric cancer patient tissues. 48

Fig. 9. The relative frequencies of LRRFIP2 splicing are significantly associated with the expression levels of ESRP1 in gastric cancer patient tissues. 50

Fig. 10. Expression levels of ESRP1 and LRRFIP2 variant 3 in gastric a cancer patents. 54

Fig. 11. Overexpression of ESRP1 reduces migration and invasion ability of gastric cancer cells and induces switching from variant 3 to variant 2. 56

Fig. 12. Exon 7 of LRRFIP2 variant 3 determines the metastatic potential of gastric cancer cells in vitro and in vivo. 60

Fig. 13. splicing switch of LRRFIP2 does not alter tumorigenicity of gastric cancer cells. 61

Fig. 14. LRRFIP2 exon 7 knockout suppresses invasion and migration a of gastric cancer cells in vitro. 63

Fig. 15. LRRFIP2 exon 7 knockout suppresses metastatic potential of a gastric cancer cells in vivo. 64

Fig. 16. Alternative splicing of LRRFIP2 regulates metastasis-associated gene network in gastric cancer cells. 70

Fig. 17. LRRFIP2 variant 2 specifically interacts with CARM1. 75

Fig. 18. LRRFIP2 variant 2 specifically interacts with CARM1. 77

Fig. 19. LRRFIP2 splicing switch determines methylase activity of a CARM1. 78

Fig. 20. LRRFIP2 variant 3 plays a critical role in regulation of a SERPINE1 expression by CARM1. 81

Fig. 21. LRRFIP2 variant 3 plays a critical role in regulation of SERPINE1 expression by CARM1. 83

Fig. 22. Knockdown of CARM1 reduces invasion and migration. 85

Fig. 23. CARM1 interacts with the p160 coactivator ACTR/SRC3/ AIB1/NCOA3 in the presence of LRRFIP2 variant 3. a Immunoprecipitation... 87

Fig. 24. CARM1 and ACTR cooperate for transcriptional regulation of SERPINE1. 89

초록보기

 선택적 스플라이싱(alternative splicing)이 다양한 암에서 비정상적으로 변해 있다는 연구결과가 꾸준히 보고되고 있지만 기능적 메커니즘은 아직 충분히 밝혀지지 않았다. 본 연구에서 우리는 epithelial splicing regulatory protein 1 (ESRP1)에 의한 leucine-rich repeat Fli-I-interacting protein 2 (LRRFIP2)의 상피성, 중간엽성 동형 단백질 스위치가 위암세포의 전이성과 밀접한 상관관계를 가지고 있음을 밝혔다. LRRFIP2의 동형단백질의 발현량과 ESRP1의 발현량이 밀접한 연관성을 가지고 있으며, 놀랍게도 LRRFIP2의 중간엽 동형 단백질의 이소성 발현을 유도할 경우 간전이가 급격하게 일어나는 현상을 관찰하였다. 또한, CRISPR/Cas9 시스템을 이용하여 LRRFIP2 동형단백질 3번의 7번째 엑손을 제거할 경우 중간엽성에서 상피성 동형단백질로 스위치가 일어나며, 이는 간전이를 유의미하게 억제시킴을 발견하였다. 메커니즘 상으로는, LRRFIP2의 상피성 동형단백질이 coactivator-associated arginine methyltransferase 1 (CARM1)과의 상호작용을 통해 CARM1의 발암성 기능을 억제함을 밝혔다. 놀랍게도, LRRFIP2의 동형단백질 3이 높게 발현된 위암환자들은 전체 생존기간도 짧은 것이 관찰되었다. 종합하자면, 본 연구는 LRRFIP2의 동형 단백질 스위치가 위암세포의 전이를 조절하는 새로운 메커니즘을 밝혔다.

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