Research On Resistance Of Hepatocellular Carcinoma To Sorafenib-targeted Therapy Based On Whole-genome Cas9

Objective:To screen the sorafenib-related key genes with sensibilization after knockout in hepatocellular carcinoma through whole-genome Cas9,further analyze the sorafenib-targeted therapy for resistant and sensitive hepatocellular carcinoma specimens using the Affymetrix whole gene expression profile chip,and obtain differentially expressed genes,and analyze the sorafenib resistance-related resistant and sensitive genes,so as to provide predictive indexes for the clinical treatment of patients with hepatocellular carcinoma with sorafenib-targeted therapy.Methods:1.Library amplification and lentiviral packaging,and exploration of pre-experimental screening conditions: The human SMMC-7721 hepatocellular carcinoma cells and mycoplasma were detected,the cell doubling time test,Puromycin screening test and IC50 test were performed,and the optimal concentration of sorafenib was determined.The optimal MOI was determined via virus infection pre-experiment.2.Establishment of susceptibility screening model and high-throughput sequencing of Cas9-sg RNA library: After amplification of hepatocellular carcinoma cell line SMMC-7721 and large-scale infection and puromycin screening,the cells were cultured with an appropriate concentration of sorafenib,and they were harvested at 0,7 and 14 d to extract the genomic DNA.After purification,the sg RNA was amplified,and the changes in sg RNA at the 3 time points were analyzed via high-throughput sequencing,and the candidate genes related to sensitivity to sorafenib were screened.3.Screening of clinical specimens: The expression profile of sorafenib-resistant and sensitive clinical hepatocellular carcinoma specimens was analyzed by Affymetrix gene chip,and differentially expressed genes in hepatocellular carcinoma tissues were screened.4.The resistant/sensitive genes closely related to the resistance of sorafenib were obtained through analyzing the sorafenib-related key genes with sensibilization after knockout in hepatocellular carcinoma through whole-genome CRISPR/Cas9 and differentially expressed genes in clinical sample gene expression profile.Results:1.A total of 1723 key drug-sensitive genes were screened in the whole genome.At 7d after drug administration(Day 7 vs.Day 0),339 key genes with sensibilization after knockout were found.A total of 1438 qualified genes were found at 14 d after drug administration(Day 14 vs.Day 0).2.1233 differentially expressed genes were obtained in sorafenib-resistant and sensitive clinical hepatocellular carcinoma specimens using Affymetrix gene chip,including 638 up-regulated ones and 595 down-regulated ones.3.The common genes were screened through analyzing the sorafenib-related key genes with sensibilization after knockout in hepatocellular carcinoma through whole-genome Cas9 and sorafenib-resistant and sensitive clinical hepatocellular carcinoma specimens using Affymetrix gene chip.At the level of hepatocellular carcinoma cells and tissues,49 common genes potentially related to drug resistant/sensitive genes were analyzed,among which CTNNB1(β-catenin),MET(c-Met/HGFR),HTATIP2,etc.may be related to resistance to sorafenib,while PRDX3,C10orf11,KCTD3,MAST4,etc.may be related to the potential target resistance of hepatocellular carcinoma to sorafenib.Its expression in hepatocellular carcinoma and clinical potential value has not been reported in the literature at present,which needs further exploration.Conclusions:It is preliminarily found that candidate genes such as CTNNB1(β-catenin),MET(c-Met/HGFR),HTATIP2,PRDX3,C10orf11,KCTD3 and MAST4 may be closely related to the resistance/sensitivity of hepatocellular carcinoma to sorafenib,which have potential important clinical value.β-catenin,c-Met and HTATIP2 are associated with the target resistance of hepatocellular carcinoma to sorafenib.The in-depth study on the genes in the target resistance of hepatocellular carcinoma to sorafenib is of great clinical significance in clarifying the mechanism of resistance of hepatocellular carcinoma to sorafenib and predictive molecular markers for effective targeted therapy.