Identification of circulating transcripts associated with adenoma-carcinoma sequence in the colorectal cancer

다국어 초록 (Multilingual Abstract)

ABSTRACT

Identification of Circulating Transcripts Associated with Adenoma-Carcinoma Sequence in the Colorectal Cancer

Li Ah KIM
Department of Biomedical Laboratory Science
The Graduate School
Yonsei University

Colorectal cancer (CRC) is the third most common cancer worldwide and the second leading cause of cancer-related mortality. The conventional model for CRC progression traces its origins from normal epithelial cells through non-advanced and advanced adenomas, ultimately culminating in cancer. While this model has been instrumental in colorectal cancer surveillance, its reliance on tissue sampling presents challenges in accurately determining cancer stages. This study addresses these limitations by identifying circulating transcriptomic biomarkers associated with the adenoma-carcinoma sequence in CRC, utilizing readily available blood samples for increased screening efficacy. Blood samples were obtained from 160 individuals scheduled for colonoscopy who were systematically divided into five groups based on both colonoscopy and histology results: CRC group, advanced adenoma (AA) group, non-advanced adenoma (NA) group, symptomatic non-disease control (NDC) group, and healthy control (HC) group. RNA sequencing was performed on 20 samples from each group, and the subsequent results were subjected to three gene ontology analysis methods. Further analysis included network examination using heat map, protein-protein interaction, and KEGG pathway analyses. The study yielded remarkable results, identifying IFI27 in the NDC group, DEFA4 in the NA group, MPO in the AA group and CD177 in the CRC group as significant circulating transcriptional biomarkers. These biomarkers, which are closely linked to immune responses, suggest the involvement of immune cells, particularly neutrophils, in CRC progression. Of particular importance is the marked upregulation of myeloperoxidase (MPO) in the NA and AA groups compared to the HC group, a validation confirmed by RT-qPCR testing on a subset of 60 blood samples. The implications of this research underscore the potential utility of specific circulating transcriptomic biomarkers associated with the adenoma-carcinoma sequence for the early detection and management of CRC. This innovative approach represents a non-invasive method to identify precancerous adenomas in the bloodstream, offering promising prospects for early intervention prior to tissue manifestation.


Key words: colorectal cancer, adenoma-carcinoma sequence (ACS), RNA sequencing, circulating transcripts, biomarker, neutrophil, MPO, RT-qPCR

국문 초록 (Abstract)

국문초록

대장암 선종-암화 기전과 관련된
순환 전사체의 식별

연세대학교 대학원
임상병리학과
김리아

대장암(CRC)은 전 세계적으로 세 번째로 흔한 암이며 암 관련 두 번째 주요 사망 원인이다. CRC 진행에 대한 선종-암화 기전은 정상 상피 세포에서 비진행성 선종과 진행성 선종을 거쳐 궁극적으로 암으로 발전하는 과정을 설명하고 있다. 이 기전은 대장암 감시에 중요한 역할을 했지만 조직 샘플링에 의존하기 때문에 실제 임상적으로 암 단계를 정확하게 결정하는데 어려움이 있다. 이 연구는 CRC의 선종-암화 기전과 관련된 순환 전사체 바이오마커를 식별하고, 암에 대한스크리닝 효능 증가를 위해 쉽게 이용 가능한 혈액 샘플을 활용함으로써 이러한 한계를 해결하고자 했다. 대장내시경 검사를 받기로 예정된 160명의 개인으로부터 혈액 샘플을 채취하여 대장내시경 검사와 조직학 결과를 바탕으로 총 5개의 그룹, CRC 그룹, 진행성 선종(AA) 그룹, 비진행성 선종(NA) 그룹, 증상이 있는 비질병 대조(NDC)그룹과 건강한 대조(HC)그룹 으로 체계적으로 분류하였다. 각 그룹당 20개 샘플, 총 100개의 샘플로 RNA sequencing을 수행하였고, 이후 결과를 3가지 유전자 온톨로지 분석 방법에 적용하였다. 추가 분석에는 히트 맵, 단백질-단백질 상호 작용 및 KEGG 경로 분석을 사용한 네트워크 연구를 시행하였다. 이 연구에서는 중요한 순환 전사체바이오마커로 NDC 그룹의 IFI27, NA 그룹의 DEFA4, AA 그룹의 MPO, CRC 그룹의 CD177을 선정할 수 있었다. 면역 반응과 밀접하게 연관되어 있는 이러한 바이오마커는 CRC 진행에 면역 세포, 특히 호중구가 관여함을 시사한다. 특히 중요한 것은 HC 그룹에 비해 NA 및 AA 그룹에서 골수과산화효소(MPO)의 현저한 상향 조절이 있는 것을 HC그룹과 NA, AA그룹에 해당하는 각 20개의 혈액 샘플, 총 60개의 혈액 샘플에 대하여 RT-qPCR 테스트를 통해 검증하였다. 혈액으로부터 순환 전사체 바이오마커를 이용하여 대장암의 진행과정을 식별하는 이러한 혁신적인 접근법은 전암성 선종을 진단하는 비침습적 방법을 제시하며, 조직에서의 암 발현 보다 앞서 혈액으로부터 순환 전사체 바이오마커를 검출함으로써 대장암에 대한 조기 진단과 예후 전략을 향상시키는데 유용함을 보여주고 있다.
________________________________________핵심되는말: 대장암, 선종-암종 서열, RNA 염기서열분석, 순환 전사체, 바이오마커, 호중구, MPO, RT-qPCR

목차 (Table of Contents)

• CONTENTS
• LIST OF FIGURES ⅳ
• LIST OF TABLES v
• ABBREVIATIONS vi
• ABSTRACT IN ENGLISH ix
• Ⅰ. INTRODUCTION 1
• Ⅱ. MATERIALS AND METHODS 12
• 1. Study subjects 12
• 2. Blood collection and RNA isolation 15
• 3. cDNA synthesis 17
• 4. Quantitative PCR assay 18
• 5. RNA sequencing analysis and differentially expressed gene analysis 19
• 6. Gene-enrichment and functional annotation analysis------------------------------- 21
• 7. Clustering heatmap analysis 22
• 8. Protein-protein interaction network analysis 24
• 9. Gene ontology analysis Ⅰ 25
• 10. Gene ontology analysis Ⅱ 26
• 11. Gene pathway analysis 27
• Ⅲ. RESULT 28
• 1. Selection process of circulating transcripts associated with the adenoma-carcinoma sequence from RNA sequencing analysis 28
• 2. Circulating transcripts differentially expressed during the transition from the healthy control group to the non-disease control group in the adenoma-carcinoma sequence 30
• 3. Circulating transcripts differentially expressed during the transition from the non-disease control group to the non-advanced adenoma group in the adenoma-carcinoma sequence 34
• 4. Circulating transcripts differentially expressed during the transition from the non-advanced adenoma group to the advanced adenoma group in the adenoma-carcinoma sequence 37
• 5. Circulating transcripts differentially expressed during the transition from the advanced adenoma group to the colorectal cancer group in the adenoma-carcinoma sequence 40
• 6. Expression patterns of circulating transcripts from the healthy control group to the colorectal cancer group in the adenoma-carcinoma sequence using clustering heatmap 47
• 7. Network patterns of circulating transcripts from the healthy control group to the colorectal cancer group in the adenoma-carcinoma sequence using protein-protein interaction 51
• 8. Gene ontology of circulation transcripts from the health control group to the colorectal cancer group in the adenoma-carcinoma sequence 54
• 9. Circulating transcripts that showed changes in the biological processes at each stage of the adenoma-carcinoma sequence 58
• 10. Activation patterns of circulating transcripts through the genetic pathway from the healthy control group to the colorectal cancer group in the adenoma-carcinoma sequence 63
• 11. Clinical validation test for two candidate biomarkers related to adenoma----- 70
• IV. DISCUSSION 72
• V. REFERENCES 84
• ABSTRACT IN KOREAN 102
•