Genetic screens in intestinal organoid (mini-gut) culture using CRISPR-Cas-9 System and studying the role of FBXL5 in colorectal cancer cells
Current in vitro research methods are often difficult to translate into successful gene(s) functional and clinical applications. Immortalised cell lines are monocellular, 2-dimensional and do not accurately encapsulate the in vivo microenvironment, while in vivo animal models are often impractical,...
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| Format: | Thesis (University of Nottingham only) |
| Language: | English |
| Published: |
2022
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| Online Access: | https://eprints.nottingham.ac.uk/68697/ |
| _version_ | 1848800508655960064 |
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| author | Jinks, Nicholas |
| author_facet | Jinks, Nicholas |
| author_sort | Jinks, Nicholas |
| building | Nottingham Research Data Repository |
| collection | Online Access |
| description | Current in vitro research methods are often difficult to translate into successful gene(s) functional and clinical applications. Immortalised cell lines are monocellular, 2-dimensional and do not accurately encapsulate the in vivo microenvironment, while in vivo animal models are often impractical, prohibitively expensive and ethically contentious. As such, novel in vitro experimental models are required to facilitate research and improve translatability, particularly in colorectal cancer, which demonstrates great genetic and phenotypic heterogeneity. Recently, methods for the in vitro culture of three-dimensional (3D) derived “organoids” have been established, which faithfully reproduce the genetic, proteomic and pharmacological characteristics of their original tissue. Organoids present the opportunity to elucidate molecular mechanisms underlying functional genetic modelling, personalised medicine and drug discovery in vitro, thereby replacing and reducing the use of in vivo animal models.
In this study, we have initially combined the CRISPR/Cas-9 genome editing system and murine intestinal/colon epithelial organoid cultures to study the functional impact of genetic knockouts of the F-box family of E3 ligases, which are poorly understood in colorectal cancer development and progression. Among the 32 F-box genes we screened, five F-box knockout murine intestinal organoid lines demonstrated differential growth pattern and morphology characteristics from healthy/normal control organoids: the fbxl5, fbxo31, fbxl18, fbxo17 and fbxl17 genes.
Moreover, we selected FBXL5 (F-box/LRR-repeat protein 5) for additional functional analysis. Previous studies reported that FBXL5 plays a key role in iron homeostasis by promoting ubiquitination and degradation of the IRP2 protein and other molecules, maintaining of hematopoietic and neural stem/progenitor cell pool, hypoxia and drug response and epithelial-to-mesenchymal transition (EMT). However, the role of FBXL5 in colorectal cancer cells is less characterised.
Furthermore, we generated CRISPR-Cas-9-mediated knockout DLD-1 and SW480 colorectal cancer cell lines to investigate the loss of FBXL5 in colorectal cancer function and activity.
We found that FBXL5 knockout significantly reduces wound healing and colony formation efficiency, and dysregulates cell morphology, epithelial-to-mesenchymal transition activity, iron homeostasis, autophagy, hypoxic cell activity and drug response in colorectal cancer cells.
In addition, toward a mechanistic approach for the characterisation of FBXL5 function and gene downstream analysis, differentially expressed genes (DEG) were identified using RNA-Seq analysis of FBXL5-knockout and parental DLD-1 colorectal cancer cells. Pathway mapping of the DEGs identified numerous novel FBXL5 roles, particularly under hypoxic conditions, including colorectal and pancreatic cancer, immune system function, spliceosome activity and carbon metabolism in cancer. However, further investigation is required to explore the significant loss of FBXL5 E3 ligase function and targeted proteins in patients with colorectal cancer.
In conclusion, we have demonstrated the value and practicality of organoids as a flexible in vitro model system for functional genetic studies, representing an important tool for improving research methodology and reducing and replacing the use of in vivo models in research. We also demonstrated that FBXL5 may be a key player in colorectal cancer progression, metastasis, and relapse via influences in iron homeostasis, autophagy, hypoxic cell activity and drug response. |
| first_indexed | 2025-11-14T20:52:41Z |
| format | Thesis (University of Nottingham only) |
| id | nottingham-68697 |
| institution | University of Nottingham Malaysia Campus |
| institution_category | Local University |
| language | English |
| last_indexed | 2025-11-14T20:52:41Z |
| publishDate | 2022 |
| recordtype | eprints |
| repository_type | Digital Repository |
| spelling | nottingham-686972022-07-31T04:41:47Z https://eprints.nottingham.ac.uk/68697/ Genetic screens in intestinal organoid (mini-gut) culture using CRISPR-Cas-9 System and studying the role of FBXL5 in colorectal cancer cells Jinks, Nicholas Current in vitro research methods are often difficult to translate into successful gene(s) functional and clinical applications. Immortalised cell lines are monocellular, 2-dimensional and do not accurately encapsulate the in vivo microenvironment, while in vivo animal models are often impractical, prohibitively expensive and ethically contentious. As such, novel in vitro experimental models are required to facilitate research and improve translatability, particularly in colorectal cancer, which demonstrates great genetic and phenotypic heterogeneity. Recently, methods for the in vitro culture of three-dimensional (3D) derived “organoids” have been established, which faithfully reproduce the genetic, proteomic and pharmacological characteristics of their original tissue. Organoids present the opportunity to elucidate molecular mechanisms underlying functional genetic modelling, personalised medicine and drug discovery in vitro, thereby replacing and reducing the use of in vivo animal models. In this study, we have initially combined the CRISPR/Cas-9 genome editing system and murine intestinal/colon epithelial organoid cultures to study the functional impact of genetic knockouts of the F-box family of E3 ligases, which are poorly understood in colorectal cancer development and progression. Among the 32 F-box genes we screened, five F-box knockout murine intestinal organoid lines demonstrated differential growth pattern and morphology characteristics from healthy/normal control organoids: the fbxl5, fbxo31, fbxl18, fbxo17 and fbxl17 genes. Moreover, we selected FBXL5 (F-box/LRR-repeat protein 5) for additional functional analysis. Previous studies reported that FBXL5 plays a key role in iron homeostasis by promoting ubiquitination and degradation of the IRP2 protein and other molecules, maintaining of hematopoietic and neural stem/progenitor cell pool, hypoxia and drug response and epithelial-to-mesenchymal transition (EMT). However, the role of FBXL5 in colorectal cancer cells is less characterised. Furthermore, we generated CRISPR-Cas-9-mediated knockout DLD-1 and SW480 colorectal cancer cell lines to investigate the loss of FBXL5 in colorectal cancer function and activity. We found that FBXL5 knockout significantly reduces wound healing and colony formation efficiency, and dysregulates cell morphology, epithelial-to-mesenchymal transition activity, iron homeostasis, autophagy, hypoxic cell activity and drug response in colorectal cancer cells. In addition, toward a mechanistic approach for the characterisation of FBXL5 function and gene downstream analysis, differentially expressed genes (DEG) were identified using RNA-Seq analysis of FBXL5-knockout and parental DLD-1 colorectal cancer cells. Pathway mapping of the DEGs identified numerous novel FBXL5 roles, particularly under hypoxic conditions, including colorectal and pancreatic cancer, immune system function, spliceosome activity and carbon metabolism in cancer. However, further investigation is required to explore the significant loss of FBXL5 E3 ligase function and targeted proteins in patients with colorectal cancer. In conclusion, we have demonstrated the value and practicality of organoids as a flexible in vitro model system for functional genetic studies, representing an important tool for improving research methodology and reducing and replacing the use of in vivo models in research. We also demonstrated that FBXL5 may be a key player in colorectal cancer progression, metastasis, and relapse via influences in iron homeostasis, autophagy, hypoxic cell activity and drug response. 2022-07-31 Thesis (University of Nottingham only) NonPeerReviewed application/pdf en cc_by https://eprints.nottingham.ac.uk/68697/1/NJ%20Thesis%20corrected%20vesion%201%2014-3.pdf Jinks, Nicholas (2022) Genetic screens in intestinal organoid (mini-gut) culture using CRISPR-Cas-9 System and studying the role of FBXL5 in colorectal cancer cells. PhD thesis, University of Nottingham. Colorectal cancer CRISPR/Cas9 organoid RNA-Sequencing F-box |
| spellingShingle | Colorectal cancer CRISPR/Cas9 organoid RNA-Sequencing F-box Jinks, Nicholas Genetic screens in intestinal organoid (mini-gut) culture using CRISPR-Cas-9 System and studying the role of FBXL5 in colorectal cancer cells |
| title | Genetic screens in intestinal organoid (mini-gut) culture using CRISPR-Cas-9 System and studying the role of FBXL5 in colorectal cancer cells |
| title_full | Genetic screens in intestinal organoid (mini-gut) culture using CRISPR-Cas-9 System and studying the role of FBXL5 in colorectal cancer cells |
| title_fullStr | Genetic screens in intestinal organoid (mini-gut) culture using CRISPR-Cas-9 System and studying the role of FBXL5 in colorectal cancer cells |
| title_full_unstemmed | Genetic screens in intestinal organoid (mini-gut) culture using CRISPR-Cas-9 System and studying the role of FBXL5 in colorectal cancer cells |
| title_short | Genetic screens in intestinal organoid (mini-gut) culture using CRISPR-Cas-9 System and studying the role of FBXL5 in colorectal cancer cells |
| title_sort | genetic screens in intestinal organoid (mini-gut) culture using crispr-cas-9 system and studying the role of fbxl5 in colorectal cancer cells |
| topic | Colorectal cancer CRISPR/Cas9 organoid RNA-Sequencing F-box |
| url | https://eprints.nottingham.ac.uk/68697/ |