Investigation of the transcriptional effects of LZTR1 in the Hep3B hepatocellular carcinoma cell line

Liver cancer is the seventh most frequently diagnosed cancer type in the world and the third leading cause of cancer-related deaths. Hepatocellular carcinoma (HCC) accounts for 85% of primary liver cancers. The LZTR1 protein, encoded by the leucine zipper-like post-translational regulator 1 (LZTR1) gene, plays a role in the reduction of RAS/mitogen-activated protein kinase (RAS/MAPK) signaling by polyubiquitinating canonical RAS and non-canonical-RIT1 proteins in the cell. LZTR1 is frequently mutated in HCC and has been found to be a tumor suppressor. Transcriptional changes induced by LZTR1 knockdown in HCC cells have not been investigated. In this thesis, we aimed to investigate the effects of LZTR1 knockout on expression changes in the Hep3B cell line. For this purpose, the LZTR1 gene was knockout by the CRISPR/Cas9 method. Comparative transcriptomic analysis of RNA samples of LZTR1 knockout and control cells aimed to determine the expression changes caused by the LZTR1 gene in Hep3B cells. For this purpose, Hep3B colonies with and without LZTR1 knockout were prepared using the CRISPR/Cas9 method. LZTR1 protein levels were analyzed using western blotting, and genomic DNA sequences were analyzed using the Sanger method to confirm LZTR1 knockout. RNA sequencing analyses revealed that knockout of LZTR1 altered the expression of 117 genes, including 20 LncRNAs, by >1.5-fold (p<0.05). Gene set enrichment analyses (GSEA) revealed that Gene Ontology (GO) terms associated with the biological processes of development and differentiation, transcription regulation, and cell signaling were enriched when the LZTR1 gene was expressed. Our validation experiments using qRT-PCR showed that the gene expressions of cadherin 11 (CDH11), tribbles pseudokinase 2 (TRIB2), and KLF transcription factor 5 (KLF5) were increased in LZTR1 knockout cells. Conversely, the gene expressions of vimentin (VIM), angiomotin-like 1 (AMOTL1), bone morphogenetic protein 7 (BMP7), and reelin (RELN) were found to be decreased. These findings are in line with the RNA sequencing results (R2= 0.96). As a result of this thesis study, data that will contribute to the elucidation of the transcriptional regulatory functions of LZTR1 in HCC were obtained and it is predicted that these findings may contribute to a better understanding of the role of LZTR1 in HCC.