Regulation of the PTEN Tumour Suppressor by its Pseudogene (PTENp1) and the MicroRNA Landscape: Potential for Future Cancer Therapy

Travis, Glena J.

Regulation of the PTEN Tumour Suppressor by its Pseudogene (PTENp1) and the MicroRNA Landscape: Potential for Future Cancer Therapy

Travis, Glena J.

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Publication Type:: Thesis
Issue Date:: 2022

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Field	Value	Language
dc.contributor.author	Travis, Glena J.
dc.date.accessioned	2023-07-14T00:57:52Z
dc.date.available	2023-07-14T00:57:52Z
dc.date.issued	2022
dc.identifier.uri	http://hdl.handle.net/10453/171494
dc.description	University of Technology Sydney. Faculty of Science.	en_US.UTF-8
dc.description.abstract	PTEN, an antagonist of the oncogenic PI3K pathway, is a well-known tumour suppressor. Subtle decreases in PTEN levels can lead to oncogenesis, thus PTEN expression is tightly regulated at multiple levels. Post-transcriptionally, PTEN is regulated by non-coding RNAs such as microRNA and the processed pseudogene of PTEN, PTENp1. microRNAs usually bind to the 3’-UTR of PTEN and decrease PTEN cellular levels. PTENp1 is expressed as sense (PTENp1-S) and anti-sense (PTENp1-AS) transcripts, both regulating PTEN. PTENp1-S shares high sequence similarity with PTEN and acts as a ceRNA to ‘sponge’ miRNAs in the shared pool with PTEN. The PTENp1-AS transcript is expressed as two isoforms; an alpha isoform that negatively regulates PTEN transcriptionally and a beta isoform that positively regulates PTEN post-transcriptionally. Various cancer and non-cancer cell lines were initially tested for expression of PTEN and both the PTENp1 sense and anti-sense transcripts to determine the prevalence of this PTEN regulatory mechanism. RT-PCR analysis showed the expression of PTEN in 9/10 cell lines tested with variable expression of the pseudogene transcripts across the cell types. Only U-2OS (osteosarcoma), HEK-293 (kidney), PNT-2 (non-cancerous prostate) and LNCaP prostate cancer cells expressed all three transcripts. Quantitation of PTEN, PTENp1-S and PTENp1-AS expression was then undertaken to determine transcript copy numbers and understand quantitative differences across the different cell lines and between cancer and non-cancerous cells. Transcript quantitation was carried out semi-quantitatively, followed by absolute quantitation using RT-qPCR and droplet digital PCR. PTEN copy number was overall higher than that of PTENp1-S and PTENp1-AS across most cell lines, except the prostate cancer cells (LNCaP and PC-3), where the pseudogene transcript levels were higher than PTEN. Additionally, pseudogene sense transcript levels decreased with prostate cancer stage (early-stage vs late-stage), confirmed using RT-qPCR and ddPCR. Previous work on another project indicated the potential for miRNA regulation of PTEN/PTENp1 through 5’-UTR interactions. Initially, microRNAs targeting the 3’-UTR, coding sequence (CDS) and 5’-UTR of PTEN and PTENp1-S were predicted using in silico bioinformatics analyses. These analyses predicted 7, 3 and 11 novel miRNAs as potential targets for the 5’UTR, CDS and 3’-UTR of PTEN and PTENp1-S, respectively. miRNA sequencing (miRNA-seq) analysis detected 29 miRNAs associated with the 5’-UTR of PTEN/PTENp1. Comparison of miRNAs detected by miRNA-seq and those predicted in silico, revealed 7 miRNAs common to both studies. These miRNAs, hsa-miR-378h, hsa-miR-6749-3p, hsa-miR-3150a-3p, hsa-miR-3180-3p, hsa-miR-497-5p, hsa-miR-370-5p and hsa-miR-195-5p represent novel PTEN and PTENp1-S 5’UTR targeting microRNA candidates for future validation.	en_US.UTF-8
dc.format	Thesis (PhD)
dc.language.iso	en_US	en_US.UTF-8
dc.relation	https://opus.lib.uts.edu.au/bitstream/10453/171494/2/02whole.pdf
dc.rights	info:eu-repo/semantics/openAccess
dc.rights	The author owns the copyright in this thesis including all reproduction and reuse rights for the work. The work may not be altered without the permission of the copyright owner. Attribution is essential when quoting or paraphrasing from this thesis.
dc.rights	© 2022 Glena J. Travis
dc.rights	au.edu.uts.lib/cph
dc.title	Regulation of the PTEN Tumour Suppressor by its Pseudogene (PTENp1) and the MicroRNA Landscape: Potential for Future Cancer Therapy	en_US.UTF-8
dc.type	Thesis
utslib.copyright.status	open_access	*

Abstract:

PTEN, an antagonist of the oncogenic PI3K pathway, is a well-known tumour suppressor. Subtle decreases in PTEN levels can lead to oncogenesis, thus PTEN expression is tightly regulated at multiple levels. Post-transcriptionally, PTEN is regulated by non-coding RNAs such as microRNA and the processed pseudogene of PTEN, PTENp1. microRNAs usually bind to the 3’-UTR of PTEN and decrease PTEN cellular levels. PTENp1 is expressed as sense (PTENp1-S) and anti-sense (PTENp1-AS) transcripts, both regulating PTEN. PTENp1-S shares high sequence similarity with PTEN and acts as a ceRNA to ‘sponge’ miRNAs in the shared pool with PTEN. The PTENp1-AS transcript is expressed as two isoforms; an alpha isoform that negatively regulates PTEN transcriptionally and a beta isoform that positively regulates PTEN post-transcriptionally. Various cancer and non-cancer cell lines were initially tested for expression of PTEN and both the PTENp1 sense and anti-sense transcripts to determine the prevalence of this PTEN regulatory mechanism. RT-PCR analysis showed the expression of PTEN in 9/10 cell lines tested with variable expression of the pseudogene transcripts across the cell types. Only U-2OS (osteosarcoma), HEK-293 (kidney), PNT-2 (non-cancerous prostate) and LNCaP prostate cancer cells expressed all three transcripts. Quantitation of PTEN, PTENp1-S and PTENp1-AS expression was then undertaken to determine transcript copy numbers and understand quantitative differences across the different cell lines and between cancer and non-cancerous cells. Transcript quantitation was carried out semi-quantitatively, followed by absolute quantitation using RT-qPCR and droplet digital PCR. PTEN copy number was overall higher than that of PTENp1-S and PTENp1-AS across most cell lines, except the prostate cancer cells (LNCaP and PC-3), where the pseudogene transcript levels were higher than PTEN. Additionally, pseudogene sense transcript levels decreased with prostate cancer stage (early-stage vs late-stage), confirmed using RT-qPCR and ddPCR. Previous work on another project indicated the potential for miRNA regulation of PTEN/PTENp1 through 5’-UTR interactions. Initially, microRNAs targeting the 3’-UTR, coding sequence (CDS) and 5’-UTR of PTEN and PTENp1-S were predicted using in silico bioinformatics analyses. These analyses predicted 7, 3 and 11 novel miRNAs as potential targets for the 5’UTR, CDS and 3’-UTR of PTEN and PTENp1-S, respectively. miRNA sequencing (miRNA-seq) analysis detected 29 miRNAs associated with the 5’-UTR of PTEN/PTENp1. Comparison of miRNAs detected by miRNA-seq and those predicted in silico, revealed 7 miRNAs common to both studies. These miRNAs, hsa-miR-378h, hsa-miR-6749-3p, hsa-miR-3150a-3p, hsa-miR-3180-3p, hsa-miR-497-5p, hsa-miR-370-5p and hsa-miR-195-5p represent novel PTEN and PTENp1-S 5’UTR targeting microRNA candidates for future validation.

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http://hdl.handle.net/10453/171494