Identification of SPP1, a cancer-associated fibroblast-derived anticancer resistance gene in hepatocellular carcinoma, and functional characterization of SORT1 as a potential driver marker for hepatocarcinogenesis
- 주제(키워드) cancer-associated fibroblast , tumor microenvironment , drug resistance , methylation , hepatocellular carcinoma
- 주제(DDC) 570
- 발행기관 아주대학교
- 지도교수 조효정
- 발행년도 2023
- 학위수여년월 2023. 8
- 학위명 박사
- 학과 및 전공 일반대학원 의생명과학과
- 실제URI http://www.dcollection.net/handler/ajou/000000032998
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
목차
Chapter 1 1
Ⅰ. INTRODUCTION 1
Ⅱ. MATERIALS AND METHODS 3
1. Isolation of fibroblasts from surgically resected liver tissues 3
2. Immunofluorescence 4
3. Collection of cell culture medium 4
4. Western blotting 4
5. Acquisition and analysis of the gene expression profiles in public omics databases 5
6. Cell culture 5
7. RNA extraction and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) 6
8. SPP1-blocking peptide and SPP1-aptamer 6
9. Establishment of sorafenib- and lenvatinib-resistant cells 7
10. Mouse tumor models 7
11. RNA-sequencing data analysis 9
12. Immunohistochemical assessment of SPP1 expression in human HCC tissues 10
13. Fluorescence quantitative analysis for protein expression of integrins 11
14. Wound healing assay 11
15. Transwell assay 12
16. Co-immunoprecipitation (co-IP) 12
17. Single-cell RNA-seq analysis 13
18. Patients and definition of clinical terms 14
19. Statistical analyses 15
Ⅲ. RESULTS 16
1. CAFs suppressed the sensitivity of HCC cells to sorafenib and lenvatinib 16
2. Identified genes specifically overexpressed in CAFs 18
3. SPP1 was overexpressed in HCC tissues from sorafenib non-responders 18
4. Selection of HCC cell lines for further functional study 19
5. CAFs were a major source of secretory SPP1 in the HCC TME 19
6. Blockade of CAF-derived SPP1 restored HCC cell sensitivity to sorafenib or lenvatinib 22
7. In vivo validation of the effect of blocking CAF-derived SPP1 on HCC resistance to TKIs 25
8. CAF-derived SPP1 bound to integrin complexes on the HCC cell surface 27
9. CAF-derived SPP1 activated the RAF/ERK/STAT3 and PI3K/AKT/mTOR signaling pathways via PKCα phosphorylation in HCC cells 28
10. SPP1-related gene signatures indicated poor prognosis in patients with HCC 31
11. CAF-derived SPP1 promoted the EMT of HCC cells 32
12. EMT-related genes were specifically expressed in SPP1-positive fibroblasts 32
13. Plasma SPP1 level prior to sorafenib or lenvatinib treatment was an independent predictor of PFS or OS in patients with advanced HCC 34
Ⅳ. DISCUSSION 41
Ⅴ. REFERENCES 44
Chapter 2 50
Ⅰ. INTRODUCTION 50
Ⅱ. MATERIALS AND METHODS 52
1. Tissue samples 52
2. Gene expression profiling 52
3. Cell culture and transfection 52
4. RNA extraction and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) 53
5. Western blotting 54
6. Expression of the SORT1 Gene at the Single-Cell Level 54
7. Cell viability and growth assay 55
8. Clonogenic proliferation assay 55
9. Wound healing assay 56
10. Mouse tumor models 56
11. Immunohistochemistry (IHC) 57
12. Apoptosis and cell cycle assays 57
13. Hoechst 33342/PI staining assay 58
14. Molecular pathway mining 58
15. Tube formation assay 59
16. Migration assay 59
17. Exploration of gene alterations in SORT1 Using cBioPortal and comparative analysis of multidimensional cancer genomics and clinical data 59
18. Quantitative methylation-specific polymerase chain reaction (qMSP) 60
19. Statistical analyses 60
Ⅲ. RESULTS 62
1. Identification of driver genes of hepatocarcinogenesis via multi-stage HCC microarray datasets 62
2. The oncogenic effect of SORT1 suppression overexpressed in HCCbiomarkers for HCC and their diagnostic performance in each cohort 69
3. The validation of the HCC progression effect of suppression SORT1 in vivo 71
4. SORT1-targeted inhibition regulates apoptosis and cell cycle arrest in HCC cells 74
5. The enrichment of angiogenesis-related genes in SORT1 and metastatic effects in vivo 77
6. Identification of mechanisms regulating overexpressed SORT1 activation in HCC 80
Ⅳ. DISCUSSION 87
Ⅴ. REFERENCES 89
목차
Ⅰ. INTRODUCTION 50
Ⅱ. MATERIALS AND METHODS 52
1. Tissue samples 52
2. Gene expression profiling 52
3. Cell culture and transfection 52
4. RNA extraction and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) 53
5. Western blotting 54
6. Expression of the SORT1 Gene at the Single-Cell Level 54
7. Cell viability and growth assay 55
8. Clonogenic proliferation assay 55
9. Wound healing assay 56
10. Mouse tumor models 56
11. Immunohistochemistry (IHC) 57
12. Apoptosis and cell cycle assays 57
13. Hoechst 33342/PI staining assay 58
14. Molecular pathway mining 58
15. Tube formation assay 59
16. Migration assay 59
17. Exploration of gene alterations in SORT1 Using cBioPortal and comparative analysis of multidimensional cancer genomics and clinical data 59
18. Quantitative methylation-specific polymerase chain reaction (qMSP) 60
19. Statistical analyses 60
Ⅲ. RESULTS 62
1. Identification of driver genes of hepatocarcinogenesis via multi-stage HCC microarray datasets 62
2. The oncogenic effect of SORT1 suppression overexpressed in HCCbiomarkers for HCC and their diagnostic performance in each cohort 69
3. The validation of the HCC progression effect of suppression SORT1 in vivo 71
4. SORT1-targeted inhibition regulates apoptosis and cell cycle arrest in HCC cells 74
5. The enrichment of angiogenesis-related genes in SORT1 and metastatic effects in vivo 77
6. Identification of mechanisms regulating overexpressed SORT1 activation in HCC 80
Ⅳ. DISCUSSION 87
Ⅴ. REFERENCES 89
