대장암에서 ECT2 유전자가 암화과정에 미치는 영향에 관한 연구

학위논문 (석사)-- 서울대학교 융합과학기술대학원 : 융합과학기술대학원 분자의학 및 바이오제약학과, 2016. 2. 김태유.Rho GTPases contribute to multiple cellular processes that could affect cancer progression, including cytoskeletal dynamics, cell cycle progression, transcriptional regulation and cell survival. One prevalent mechanism involves aberrant Rho activation via the deregulated expression and/or activity of Rho family guanine nucleotide exchange factors (RhoGEFs). Here, we aim to identify RhoGEFs that mediate colorectal cancer progression. Using a TCGA transcriptome data, we identified that ECT2 (epithelial cell transforming sequence 2), one of the RhoGEFs family, is more over-expressed in colorectal cancer than in adjacent normal tissue. We also confirmed this by using quantitative real-time PCR. Knockdown of ECT2 by siRNA regulates the activity of RHOA but not CDC42 and RAC1. Inhibition of ECT2 suppresses cell migration, invasion and proliferation in colorectal cancer cell lines via regulating RHOA/AKT pathway. In addition, we investigated the therapeutic use of ECT2. ECT2 is down-regulated by chemotherapy drugs like oxaliplatin, irinotecan, 5-FU in p53 wild type cell lines. Conversely in p53 mutant cell lines, ECT2 is up-regulated. Knockdown of ECT2 and chemotherapy alone generally inhibited cell proliferation. However, in p53 mutant cell line, the combination of siRNA ECT2 with chemotherapy had more effect on the inhibition of cell proliferation. This study suggests that ECT2 could be a potential biomarker and a therapeutic target for treating colorectal cancer. Also that combination of siRNA ECT2 with chemotherapy could be a novel molecular therapy for the patients with p53 mutation.Introduction 1 Material and Methods 3 Cell culture and siRNA Transfection 3 cDNA synthesis and qRT PCR 3 Reagents 4 Western Blotting 4 RHOA, CDC42 and RAC1 pull down assay 5 Wound healing assay 6 In vitro invasion assay 6 Cell proliferation assay 7 Results 8 1. Analysis of Rho GEF expression profiles using mRNA datasets 8 2. Up-regulation of ECT2 expression in colorectal cancer and investigation of ECT2 protein expression level in colorectal cancer cell lines 9 3. siRNA-Mediated inhibition of Ect2 decreased colorectal cancer cell Migration and Invasion 10 4. siRNA-Mediated inhibition of ECT2 suppressed cell proliferation of colorectal cancer 11 5. Silencing the expression of ECT2 inhibits RHOA / AKT signaling pathway 12 6. Chemotherapy drug regulated the expression of ECT2 in CRC 14 7. Chemotherapy combined with siRNA ECT2 inhibited cellular proliferation more effective in p53 mutant cells 15 Discussion 37 References 40 Abstract in Korean 44Maste