Screening Of Cisplatin Resistance-related Genes In Oral Squamous Cell Carcinoma Using CRISPR/Cas9 Library And RNA-Seq Technology

ObjectiveAccording to the screening method of crispr-cas9 library,human Gec KOv2(A)library was used to conduct genome-wide cisplatin resistance gene screening on CAL27 cells.At the same time,a strain of CAL27/Cisplatin cells resistant to Cisplatin was screened and cultured by increasing drug concentration in vitro,and the differences in mRNA expression profiles of CAL27/Cisplatin and CAL27 cells in each group were detected and analyzed after normal culture and Cisplatin treatment.CRISPR/Cas9 library data of high-throughput screening and transcriptome sequencing data for joint bioinformatics analysis,finally selected in CAL27 cells cisplatin resistance mechanisms play an important role in the key genes,and verify its function and related mechanism,for further study of Oral Squamous Cell Carcinoma cisplatin resistance mechanisms and development of cisplatin resistance reversal drugs to provide important basis.Methods1.Human Gec KOv2(A)library virus carrying puromycin resistance was infected into CAL27 cells,and puromycin resistance was selected to infect positive cells to obtain stable transfected cells.Then give the solvent DMSO and the drug cisplatin for positive screening to extract the g DNA of viable cells.PCR amplify the target fragments containing g RNA,high-throughput sequencing the changes of abundance of g RNA in each group,and bioinformatics analysis to determine the significant difference of cisplatin resistance-related genes in CAL27 cells.2.CAL27/Cisplatin cell lines were screened and cultured by the method of increasing concentration in vitro.Transcriptome sequencing was performed on CAL27 cells,CAL27/Cisplatin cells and CAL27/Cisplatin cells in normal culture.3.CRISPR/Cas9 high-throughput screening data and transcriptome sequencing data were combined for analysis to finally identify the key genes that play an important role in the mechanism of cisplatin resistance in CAL27 cells.4.Preliminary function and mechanism verification of the selected key genes were conducted through CAL27 cells and animal experiments.Results1.A total of 112 candidate genes related to cisplatin resistance in CAL27 cells were obtained through the CRISPR/Cas9 high-throughput screening.Among them,13 candidate genes related to cisplatin tolerance are ERCC8,SMEK1,PVRL3,PPP4R2,UVSSA,ZNF98,SLFN12 L,PCDHGB2,HTRA1 and C12orf42,and 99 candidate genes related to cisplatin sensitivity are PMVK,EXOSC3,DHRSX＿X,PPP1R35,C16orf59,ATP5 I,C18orf21,AASDHPPT,IRF7 and NMD3.2.CAL27 /Cisplatin cell lines were screened by in vitro concentration increment method and successfully cultured.3.Through transcriptome sequencing screening,a total of 25 candidate genes related to cisplatin resistance were obtained.All the 25 candidate genes showed relatively low expression in CAL27/Cisplatin cells and relatively high expression in CAL27 cells after Cisplatin treatment,while there was no significant difference between CAL27/Cisplatin and CAL27 cells in normal culture.4.Combined analysis of the CRISPR/Cas9 high-throughput screening data and transcriptome sequencing data showed that the IRF7 gene is a key gene of CAL27 cells that plays an important role in the mechanism of cisplatin resistance.5.Cell function experiments and animal experiments have preliminarily proved that IRF7 knockdown can reduce the sensitivity of CAL27 cells to cisplatin and produce cisplatin resistance.ConclusionsIRF7 gene is a key gene that plays an important role in the mechanism of cisplatin resistance in CAL27 cells,and knockdown of IRF7 expression can reduce the sensitivity of CAL27 cells to cisplatin and generate cisplatin resistance.Therefore,IRF7 is expected to be a marker for predicting human OSCC cisplatin resistance and a potential therapeutic target for reversing human OSCC cisplatin resistance.