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Identification and functional analysis of a circular rna circznf800 involved in regulating colorectal cancer stemness properties

Colorectal cancer (CRC) is one of the most diagnosed cancers. Effective therapeutic interventions remain challenging due to the existence of a rare population of chemoresistant CRC cancer stem cells (CrCSC). In a spheroidal culture model, the CrCSC population is enriched with enhanced CSC-like prope...

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Main Author: Vimalan, Rengganaten
Format: Final Year Project / Dissertation / Thesis
Published: 2023
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R Medicine (General)
Online Access:http://eprints.utar.edu.my/5535/1/1706744%2DVIMALAN.pdf
http://eprints.utar.edu.my/5535/
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spelling my-utar-eprints.55352023-06-30T08:17:20Z Identification and functional analysis of a circular rna circznf800 involved in regulating colorectal cancer stemness properties Vimalan, Rengganaten R Medicine (General) Colorectal cancer (CRC) is one of the most diagnosed cancers. Effective therapeutic interventions remain challenging due to the existence of a rare population of chemoresistant CRC cancer stem cells (CrCSC). In a spheroidal culture model, the CrCSC population is enriched with enhanced CSC-like properties. However, the molecular events governing CSC properties in the CrCSC population remains poorly understood. Circular RNAs (circRNAs) are long non-coding regulatory RNAs associated with various biological functions. The present study aimed to elucidate the biological role of circRNAs in maintaining CSC properties in CRC cells. CircRNA sequencing was performed using Illumina technology. qRT-PCR and RNA FISH were used to evaluate the expression levels of circRNA. In vitro transcription and CRISPR Cas13d RNA editing system were used to modulate the expression levels of the circRNA. Whole-transcriptome sequencing of RNA of CRC spheroidal cells revealed over 8,000 differentially expressed circRNAs. The top 8 differentially expressed circRNAs were identified and the interacting miRNAs and mRNA transcripts were predicted. KEGG analysis revealed the enrichment of various CSC-associated signalling pathways; the pluripotency regulating pathways were most significantly up-regulated. Two circRNAs, hsa_circ_0066631 (circDCBLD2) and hsa_circ_0082096 (circZNF800), were predicted and experimentally validated to sponge five miRNAs, releasing posttranscriptional inhibition of six mRNA transcripts involved in the regulation of stemness. Literature analysis showed that the predicted miRNAs acted as tumour suppressors and the targeted transcripts regulated various CSC signalling pathways. As a candidate circRNA for further in-depth analysis, circZNF800 was shown to be significantly up-regulated in CRC tumour tissues. CircZNF800 overexpression promoted CSC phenotypes, including proliferation, expression of CSC and intestinal stem cell markers, and tumorigenicity, all of which were reversed by CRISPR Cas13d-mediated circZNF800 knockdown. CircZNF800 was shown to sponge miR-140-3p, miR-382 and miR-579 which, in turn, increased the levels of ALK7, FZD3 and WNT5A, which are involved in the regulation of CSC properties. In conclusion, circZNF800, identified in CrCSC circRNA profiling, was shown to participate in regulating CSC properties in CRC. 2023 Final Year Project / Dissertation / Thesis NonPeerReviewed application/pdf http://eprints.utar.edu.my/5535/1/1706744%2DVIMALAN.pdf Vimalan, Rengganaten (2023) Identification and functional analysis of a circular rna circznf800 involved in regulating colorectal cancer stemness properties. PhD thesis, UTAR. http://eprints.utar.edu.my/5535/
institution Universiti Tunku Abdul Rahman
building UTAR Library
collection Institutional Repository
continent Asia
country Malaysia
content_provider Universiti Tunku Abdul Rahman
content_source UTAR Institutional Repository
url_provider http://eprints.utar.edu.my
topic R Medicine (General)
spellingShingle R Medicine (General)
Vimalan, Rengganaten
Identification and functional analysis of a circular rna circznf800 involved in regulating colorectal cancer stemness properties
description Colorectal cancer (CRC) is one of the most diagnosed cancers. Effective therapeutic interventions remain challenging due to the existence of a rare population of chemoresistant CRC cancer stem cells (CrCSC). In a spheroidal culture model, the CrCSC population is enriched with enhanced CSC-like properties. However, the molecular events governing CSC properties in the CrCSC population remains poorly understood. Circular RNAs (circRNAs) are long non-coding regulatory RNAs associated with various biological functions. The present study aimed to elucidate the biological role of circRNAs in maintaining CSC properties in CRC cells. CircRNA sequencing was performed using Illumina technology. qRT-PCR and RNA FISH were used to evaluate the expression levels of circRNA. In vitro transcription and CRISPR Cas13d RNA editing system were used to modulate the expression levels of the circRNA. Whole-transcriptome sequencing of RNA of CRC spheroidal cells revealed over 8,000 differentially expressed circRNAs. The top 8 differentially expressed circRNAs were identified and the interacting miRNAs and mRNA transcripts were predicted. KEGG analysis revealed the enrichment of various CSC-associated signalling pathways; the pluripotency regulating pathways were most significantly up-regulated. Two circRNAs, hsa_circ_0066631 (circDCBLD2) and hsa_circ_0082096 (circZNF800), were predicted and experimentally validated to sponge five miRNAs, releasing posttranscriptional inhibition of six mRNA transcripts involved in the regulation of stemness. Literature analysis showed that the predicted miRNAs acted as tumour suppressors and the targeted transcripts regulated various CSC signalling pathways. As a candidate circRNA for further in-depth analysis, circZNF800 was shown to be significantly up-regulated in CRC tumour tissues. CircZNF800 overexpression promoted CSC phenotypes, including proliferation, expression of CSC and intestinal stem cell markers, and tumorigenicity, all of which were reversed by CRISPR Cas13d-mediated circZNF800 knockdown. CircZNF800 was shown to sponge miR-140-3p, miR-382 and miR-579 which, in turn, increased the levels of ALK7, FZD3 and WNT5A, which are involved in the regulation of CSC properties. In conclusion, circZNF800, identified in CrCSC circRNA profiling, was shown to participate in regulating CSC properties in CRC.
format Final Year Project / Dissertation / Thesis
author Vimalan, Rengganaten
author_facet Vimalan, Rengganaten
author_sort Vimalan, Rengganaten
title Identification and functional analysis of a circular rna circznf800 involved in regulating colorectal cancer stemness properties
title_short Identification and functional analysis of a circular rna circznf800 involved in regulating colorectal cancer stemness properties
title_full Identification and functional analysis of a circular rna circznf800 involved in regulating colorectal cancer stemness properties
title_fullStr Identification and functional analysis of a circular rna circznf800 involved in regulating colorectal cancer stemness properties
title_full_unstemmed Identification and functional analysis of a circular rna circznf800 involved in regulating colorectal cancer stemness properties
title_sort identification and functional analysis of a circular rna circznf800 involved in regulating colorectal cancer stemness properties
publishDate 2023
url http://eprints.utar.edu.my/5535/1/1706744%2DVIMALAN.pdf
http://eprints.utar.edu.my/5535/
_version_ 1770556050345295872
score 13.211869

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