Dissecting the role of UBC gene knockout in Gastric Cancer: Insights from CRISPR/Cas9 experiments / Analisi del ruolo del knockout del gene UBC nel cancro gastrico: approfondimenti da esperimenti con sistema CRISPR/Cas9.

Abstract This thesis investigates the role of polyubiquitin gene UBC in gastric cancer (GC) using CRISPR/Cas9-mediated UBC exon 2 knockout in primary (23132/87) and metastatic (KATO III) gastric cancer cell lines. Initial knockout validation experiments were performed in HEK293T cells to ensure reliable gene editing outcome. Following confirmation of efficient gene editing, CRISPR/Cas9-mediated UBC knockout was conducted in both GC cell lines, followed by the selection of single-cell isolated clones for further investigation. Our findings revealed a homozygous UBC knockout in the primary cell line, and a heterozygous knockout in the metastatic cell line, indicating distinct responses to UBC gene disruption. The impact of UBC knockout in the two GC cell lines was evaluated. Our results show that UBC gene knockout significantly lowered the cell proliferation and migration rates, along with a reduced cell viability in stress challenged primary gastric cancer cells, highlighting an important role of UBC in their survival. The heterozygous UBC knockout detected in KATO III suggests a potential sensitivity of metastatic cells to complete UBC depletion, revealing new insights into the dependency of these cells on ubiquitin genes for survival. Our research highlighted the UBC gene as a potential therapeutic target in primary gastric cancer. Further research focusing on UBC's role in survival mechanisms may pave the way for novel therapeutic strategies in managing gastric cancer.

This thesis investigates the role of polyubiquitin gene UBC in gastric cancer (GC) using CRISPR/Cas9-mediated UBC exon 2 knockout in primary (23132/87) and metastatic (KATO III) gastric cancer cell lines. Initial knockout validation experiments were performed in HEK293T cells to ensure reliable gene editing outcome. Following confirmation of efficient gene editing, CRISPR/Cas9-mediated UBC knockout was conducted in both GC cell lines, followed by the selection of single-cell isolated clones for further investigation. Our findings revealed a homozygous UBC knockout in the primary cell line, and a heterozygous knockout in the metastatic cell line, indicating distinct responses to UBC gene disruption. The impact of UBC knockout in the two GC cell lines was evaluated. Our results show that UBC gene knockout significantly lowered the cell proliferation and migration rates, along with a reduced cell viability in stress challenged primary gastric cancer cells, highlighting an important role of UBC in their survival. The heterozygous UBC knockout detected in KATO III suggests a potential sensitivity of metastatic cells to complete UBC depletion, revealing new insights into the dependency of these cells on ubiquitin genes for survival. Our research highlighted the UBC gene as a potential therapeutic target in primary gastric cancer. Further research focusing on UBC's role in survival mechanisms may pave the way for novel therapeutic strategies in managing gastric cancer.This thesis investigates the role of polyubiquitin gene UBC in gastric cancer (GC) using CRISPR/Cas9-mediated UBC exon 2 knockout in primary (23132/87) and metastatic (KATO III) gastric cancer cell lines. Initial knockout validation experiments were performed in HEK293T cells to ensure reliable gene editing outcome. Following confirmation of efficient gene editing, CRISPR/Cas9-mediated UBC knockout was conducted in both GC cell lines, followed by the selection of single-cell isolated clones for further investigation. Our findings revealed a homozygous UBC knockout in the primary cell line, and a heterozygous knockout in the metastatic cell line, indicating distinct responses to UBC gene disruption. The impact of UBC knockout in the two GC cell lines was evaluated. Our results show that UBC gene knockout significantly lowered the cell proliferation and migration rates, along with a reduced cell viability in stress challenged primary gastric cancer cells, highlighting an important role of UBC in their survival. The heterozygous UBC knockout detected in KATO III suggests a potential sensitivity of metastatic cells to complete UBC depletion, revealing new insights into the dependency of these cells on ubiquitin genes for survival. Our research highlighted the UBC gene as a potential therapeutic target in primary gastric cancer. Further research focusing on UBC's role in survival mechanisms may pave the way for novel therapeutic strategies in managing gastric cancer.