CRISPR/Cas9 Screening And Identification Of New Everolimus Resistance Target MAGIX In Renal Cell Carcinoma

ObjectiveClinically,mTOR inhibitors are widely used in the treatment of various tumors,especially everolimus in the treatment of metastatic renal cell carcinoma.However,in clinical practice,there is a large individual difference in efficacy,and drug resistance is common,which greatly limits its efficacy.In the mechanism of resistance to mTOR inhibitors,genetic changes in tumors play an importance part in drug-resistance.The existing related researches are mostly limited to the screening of drug-resistance-related gene targets from the limited number of cases.There is still lack of evaluation from the whole genome level for mTOR inhibitor-resistance related genes.The development and application of CRISPR/Cas9 technology provide tremendous convenience for gene function research.Here,we use the CRISPR/Cas9 library screening method to find mTOR inhibitor resistance related targets at the whole genome level,then perform functional verification and clinical correlation analysis in order to find a reversal mTOR inhibitor resistance target and strategy.Methods1.The CRISPR/Cas9 screening model of everolimus-resistance target for renal cell carcinoma was established through plasmid library amplification,virus library packaging,screening conditions preparation,and drug resistance screening process,then high-throughput sequencing was performed.2.A series of bioinformatics methods such as MageCK were used to carry out quality control and analysis of sequencing data,combined with RNA-seq of everolimus-resistance A498 cell line,servaval potential drug resistance targets were found.3.Using CRISPR knockout,plasmid transfection methods,combined with drugs sensitivity experiment,plate cloning experiment,and apoptosis experiments,for functional verification of drug resistance target.4.Cryopreserved tissue specimens of patients with renal cancer undergoing postoperative administration of everolimus were used to verify the correlation between the expression level of the potential resistant target and clinical efficacy by real-time quantitative PCR.5.Using the method of tissue chip,combined with clinical database data,analyze the clinical value of drug resistant target in evaluating the prognosis of overall population renal cell carcinoma patients.ResultsThrough CRISPR/Cas9 drug resistance gene screening,bioinformatics analysis,correlation analysis of clinical efficacy,and drugs sensitivity experiment,MAGIX was found to mediate everolimus resistance in renal cell carcinoma.In further functional experiments,compared to normal renal cell carcinoma cells,we found that after MAGIX knockout,cell colony forming ability was significantly enhanced and the proportion of apoptotic cells was significantly reduced under the same concentration of everolimus.At the same time,tissue microarray analysis revealed that the lower of MAGIX expression level indicated the worse prognosis in the overall population of patients with renal cancer,and this conclusion was consistent with the results of the TCGA database analysis.ConclusionThe expression levels of MAGIX were closely related to the resistance of everolimus.The low expression level of MAGIX could be used as a biomarker to guide individualized treatment of renal cell carcinoma.In addition,the expression level of MAGIX correlated with the overall survival of patients with renal cell carcinoma.The low expression level of MAGIX indicated a shorter overall survival time,which can be used as a novel molecular marker for predicting the prognosis of renal cancer.