Identification of gene(s) associated with pancreatic cancer metastasis by genome-wide CRISPR/Cas9 screening

Ph.D. ; Background and Aims: Pancreatic cancer is one of the most lethal cancer. The major cause of its high mortality is the metastasis nature of pancreatic cancer. After decades of efforts, several studies have supported the hypothesis that pancreatic cancer originated from consecutively accumulating of gene mutations. However, genes regulated the metastasis of pancreatic cancer are largely unknown. The difficulty in studying cancer metastasis is ascribed to the extreme complex metastasis cascades. CRISPR/Cas9 becomes a powerful genome-wide screening tool to study biological processes. Here we took advantage of the orthotopic implantation model and CRISPR/Cas9 system to identify the genes that associated with pancreatic cancer metastasis. We studied how these genes regulated the metastasis of pancreatic cancer in vitro and in vivo and further dissected its possible mechanisms underneath. ; Methods: We established mutated pancreatic cancer cell pool by lentivirus of CRISPR/Cas9 genome-wide sgRNA library. We implanted the mutated cell pool intothe pancreas of nude mice and sequenced sgRNA motifs in the metastasis tumors to identify the hit genes which may promote tumorigenesis and metastasis. We analyzed the public clinical datasets (GEO, TCGA) to reveal the relevance of these genes to the clinical significance and measured their expression in pancreatic cancer cell lines and clinical samples. We manipulated candidate genes in pancreatic cancer cell lines by knocking out, or rescued, or over expressed, then we conducted the CCK-8, colony formation, soft agar, wound healing and Boyden chamber assays and orthotopic implantation to confirm their functions in cell proliferation, survival, migration, and invasion in vitro and metastasis in vivo. We used epigenetic regulation drugs,knockdown or overexpression experiments and chromatin immunoprecipitation assays to illustrate how the expression of the candidate gene was regulated by epigeneticmechanism in pancreatic cancer. ; Results: We identified some genes which ...