CRISPR-cas9 Screening Identified Lethal Genes Enriched in Hippo Kinase Pathway and of Prognosis Significance in primary Low-grade Glioma

Abstract

Abstract Background Lethal genes in low-grade gliomas (LGGs) may have an essential prognostic significance and therefore need to be systematically analyzed. The purpose of this study is to analyze essential genes correlated with cell survival by evaluating CRISPR-cas9 screening data, leading to the identification of novel treatment targets for patients. Methods In this study, genes related to cell viability from the Depmap CRISPR-cas9 screen were intersected to differentially expressed genes (DEGs) between tumor and GTEx normal tissues from TCGA. The LASSO regression method was used to construct a signature that used to anticipate overall survival in patients with LGG. An evaluation of the signature was carried out using both multivariate and univariate Cox regression. Then, we determined which key pathways were modulated by this signature by comparing DEGs between low- and high-risk patients. WGCNA was conducted to identify modules associated with high-risk. In this study, we also performed enrichment analysis to identify pathways mediated by DEGs, overlapping genes, and genes shared in the WGCNA. Finally, we used to western blot, qRT-PCR and IHC to detect the expression of hub genes. Results Using the TCGA database, 145 oncogenes were identified as overexpressed. These genes were intersected with lethal genes identified in the Depmap database, which are enriched in Hippo pathways. A total of 19 genes were used to construct the gene signature by means of LASSO regression. The median risk score (0.752324) was employed to distinguish between low- and high-risk patients. The patients with high-risk characteristics showed a shorter OS duration in the internal training, the internal validation, and the external validation datasets. Ultimately, the Hippo signaling pathway was the predominantly enriched pathway in 145 genes, DEGs, and 3 modular genes in WGCNA. Finally, we found differences of hub genes expression in different clinical samples. Importantly, protein and mRNA expression of REP65 was significantly up-regulated in tumor cells both in the public cohort and our cohort. Conclusion The hippo signaling pathway detected based on CRISPR-cas9 screening is a critical regulator of viability and tumor proliferation and therefore is an innovative new target for treating cancerous brain tumors, including LGG.