Study On The Effect Of S100A8 Gene On Invasion And Migration Of Nasopharyngeal Carcinoma Cells Based On CRISPR/Cas9 Technique

Objective and Contents:In this study,the effects of S100A8 gene on proliferation,invasion and migration of nasopharyngeal carcinoma(NPC)cells were studied by using CRISPR/Cas9 technique to construct CNE2 cell line with S100A8 gene knockout.The main contents of the study include three parts: 1.The stable transformation cell line of nasopharyngeal carcinoma(NPC)with Cas9 protein was constructed by lentivirus transfection.Through plasmid transfection,the gRNA targeting S100A8 gene was inserted into the cell line obtained from the previous step,and the S100A8 gene was knockout.To investigate the effect of S100A8 knockout on invasion and migration of nasopharyngeal carcinoma(NPC)cells.Methods:1.By lentivirus transfection,CNE1 cells and CNE2 cells were transfected with appropriate moi values.After transfection,the cells were screened with appropriate concentration of(BSD),and theresistant CNE1,to BSD was collected.CNE2 cells were cultured.The expression of Cas9 gene and protein in CNE1 cells and CNE2 cells without lentivirus infection were detected by qRT-PCR test and Western Blot test to verify the success of the construction of Cas9 protein stable cell line of nasopharyngeal carcinoma(NPC).2.The gRNA sequence of S100A8 gene's first and second exons was designed by gRNA online design website.The LentiGuide-Puro plasmid vector was digested by Esp3 I Fast Digest,and the digested product was obtained by agarose gel electrophoresis and gel recovery.The gRNA was ligated to the LentiGuide-Puro vector.The vector targeting S100A8 gene was constructed and the recombinant plasmid was transfected into nasopharyngeal carcinoma cells with stable expression of Cas9 protein by lipofectamine transfection.The S100A8 gene was knockout by Cas9 protein under the guidance of gRNA.The knockout results were verified by Western Blot.3.Whether or not the migration ability of CNE2 cells was affected by S100A8 knockout was detected by cell scratch test and Transwell migration assay.The invasive ability of CNE2 cells after S100A8 gene knockout was detected by Transwell invasion assay.The mRNA expression of(MMPs),a matrix metalloproteinases associated with invasion and migration,was detected by qRT-PCR in CNE2 cells after knockout of S100A8 gene.Results:1.The results of qRT-PCR showed that both lentivirus-transfected CNE1 cells and CNE2 cells expressed Cas9 protein gene,but untransfected CNE1 cells and CNE2 cells did not express Cas9 protein gene.The results of Western Blot showed that Cas9 protein could not be expressed in CNE1 cells and CNE2 cells without lentivirus transfection,but Cas9 protein could be expressed in both CNE1 cells and CNE2 cells.2.The results of agarose gel electrophoresis showed that the correct linearized fragment of LentiGuide-Puro was obtained by Esp3 I double site digestion,and the results of PCR and sequencing showed that gRNA was successfully ligated into LentiGuide-Puro plasmid vector.The results of Western Blot showed that,in CNE2 cells without any treatment,the CNE2 cell lines stably transfected with Cas9 protein and CNE2 cells stably transfected with Cas9 protein transfected with LentiGuide-Puro blank plasmid were all expressed by S100A8 gene,and the expression of S100A8 gene in CNE2 cells transfected with LentiGuide-Puro blank plasmid was found in the CNE2 cell lines stably transfected with Cas9 protein obtained in previous experiments.However,CNE2 cells stably transfected with Cas9 protein transfected with LentiGuide-Puro-gRNA recombinant plasmid no longer expressed S100A8 gene.3.The results of cell scratch test showed that the migration area of CNE2 cells after S100A8 gene knockout was significantly lower than that of untreated CNE2 cells,and the ability to penetrate the membrane of CNE2 cells after S100A8 gene knockout decreased obviously from the results of Transwell migration test,and the migration area of CNE2 cells after S100A8 gene knockout was significantly lower than that of untreated CNE2 cells.The results of Transwell invasion test showed that the invasiveness of CNE2 cellswas inhibited after S100A8 knockout.At the gene level,the mRNA expression of metalloproteinases MMP3,MMP7,MMP12,a mechanism related to invasion and migration,was down-regulated after S100A8 knockout.Conclusions:Compared with the normal nasopharyngeal carcinoma poorly differentiated cell CNE2,the invasion and migration ability of nasopharyngeal carcinoma cells after S100A8 gene knockout was significantly reduced.