Genome-wide CRISPR/Cas9 Screening Identify Genes Mediating Regorafenib Resistance In Hepatocellular Carcinoma

Liver cancer is the sixth most common cancer world wild.Because of the high malignance,limited treatments,and frequent drug-resistance,liver cancer is the third most deadly tumor.Regorafenib is a multi-kinase inhibitor which has one more fluorine atom than sorafenib in structure,but it has better pharmacological activity.Clinically,regorafenib is often used in patients with liver cancer who have low effectiveness in sorafenib treatment.However,in clinical practice,there are large individual differences in the efficacy of regorafenib,and drug resistance is widespread,which greatly limits its effectiveness.Although studies have shown that the genetic changes of tumors play an important role in the resistance to regorafenib,the specific genes and mechanism is not clear,and more systematic and in-depth research is needed.This work identified genes involved in regorafenib resistance in liver cancer cells by using genome-wide CRIPSPR/Cas9 knock out library screening,then tested functions of the identified potential genes in liver cancer cells,and analyzed the potential molecular mechanism under regorafenib resistance.These studies identified genes and mechanism involved in regorafenib resistance,which provides a theoretical basis for overcoming regorafenib resistance in liver cancer.Firstly,in this project the GeCKO v2.0 human library which targets 19,050 genes was used to transfect HepG2-Cas9 cells for screening,combined with high-throughput sequencing technology,and the enrichment and difference analysis were performed.There are 9 genes were enriched in regorafenib-resistant cells compared to control cells.Furthermore,5 of the 9 genes,SHISA3,RRBP1,ARHGAP35,CSK,and ANO1 are low expression in regorafenib-resistant cells.Then the potential 5 genes were knocked down in liver cancer cells to test cell sensitivity to regorafenib,and ARHGAP35 was identified as the only candidate gene participated in drug resistance.And the study found that gene ARHGAP35 can regulate apoptosis and migration-related signaling pathways in liver cancer cells,which may be involved in the resistance of regorafenib.