Author: Chiu, Hiu Ching
Title: Hypothesis driven loss-of-function screen in mice for cooperating genes involved with the canonical WNT signaling molecular class of hepatocellular carcinoma
Advisors: Keng, Vincent (ABCT)
Degree: M.Phil.
Year: 2023
Subject: Liver -- Cancer -- Genetic aspects
Hong Kong Polytechnic University -- Dissertations
Department: Department of Applied Biology and Chemical Technology
Pages: 140 pages : color illustrations
Language: English
Abstract: Hepatocellular carcinoma (HCC) is the major form of primary liver cancer globally. Due to the divergency in the genetic background, different molecular subclasses of HCC were identified, in which the beta-catenin (CTNNB1) subclass was found to be affecting around 40% of HCC patients. CTNNB1 is the central molecule of the WNT signaling pathway, and its mutation is proven to promote HCC. As tumour development and progression involve multiple gene interactions, we are interested in whether there are any genes that work with CTNNB1 together to promote HCC.
In order to address this research interest, the CRISPR-Cas9 system and the Sleeping Beauty (SB) transposon system were used in the forward genetic screen in the FSE transgenic mouse model, where the mice have the ability to express Cas9 nuclease and SB transposase to knockout protein coding genes via specific gRNA sequences and overexpress CTNNB1 simultaneously.
By sequencing the tumour and normal liver samples from the experimental mice, Neurofibromin 2 (NF2) and Proline rich mitotic checkpoint marker (PRCC) were chosen for further validation of their roles in CTNNB1-associated HCC based on the degree of gRNA hit count, clinical relevance and novelty. NF2 is a tumour suppressor gene well-known for its involvement in the Hippo singling pathway to cause HCC. Meanwhile, PRCC is a novel gene with little known functions.
In vitro validation for NF2 was conducted in the SNU-449 and HHL7 cell lines. Knockout of NF2 in these cell lines showed increased cell proliferation and wound healing rate, with the support of up-regulation of CTNNB1, MYC, MMP2 and SNAI1. RNA sequencing was performed for the NF2 knockout cell lines and Endoglin (ENG) were identified as a potential gene involved in this HCC mechanism. qPCR results confirmed that ENG was up-regulated in NF2 knockout cells, confirming the knockout of NF2 lead to the increased ENG level and reduced SERPINF1, SFRP1 and SOX2 levels to promote WNT and therefore lead to HCC development.
In vivo reverse screen was performed in the FSE transgenic mice by injecting Nf2 targeting gRNA and CTNNB1 overexpression plasmids into the mouse liver. qPCR results confirmed that Nf2 was successfully down-regulated, with Ctnnb1 and Myc up-regulation observed at the same time. The Nf2 knockdown mice also showed increased Eng level and reduced Serpinf1, Sfrp1 and Sox2 levels, which is consistent with the in vitro results.
The role of PRCC in HCC remains investigative. The overexpression of PRCC could promote cell proliferation and migration in certain cell lines. As for the in vivo reverse screen for Prcc, the knockout was not successful and therefore no significant differences were observed in tumour formation and gene expression.
Taken together, this project contributed to how NF2 is involved in the CTNNB1-associated HCC by showing that its down-regulation could increase ENG level to promote HCC development via the WNT signaling pathway.
Rights: All rights reserved
Access: open access

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