CRISPR/Cas9-mediated Apoptosis Of Nasopharyngeal Carcinoma Cells Via Targeting Epstein-barr Virus(EBV)

Objective:First,established a stable CNE-2 cells express CRISPR/ Cas9 gRNA cooperate Cas9 can directly precise cleavage in a predefined target region LMP1 gene and so down LMP1 expression,and foreign genes have been successf?lly used to replace the target gene HDR-mediated gene replacement seamless EBV infected cells CNE-2.CRISPR/Cas9 significantly inhibited the proliferation of EBV contrast,the CRISPR/Cas9 system inhibitors reduce the reduce of LMP1 and EBV proliferation,studies have assessed the LMP1 CNE-2 of LMP1 mediated the influence of cell growth reg?lation,LMP1 expression of CNE-2 cells growth has a positive role in reg?lating,CRISPR/Cas9 mediated LMP1 gene knockout decreased significantly the CNE-2 cell reg?lation.Methods:The target sequence was designed by using Crispr online query tool,and EBNA1-f,EBNA1-r,LMP1-f and LMP1-R were compared to select the target gene sequences of EBV.Then establish CRISPR/Cas9 basis gRNA,the EBNA1 and connect with EBV LMP1 gene,get the CRISPR/Cas9 systems,plasmid extraction CRISPR/Cas9 plasmid extraction kit,and then save to 20 degrees.CRISPR/Cas9 exogenous shear activity detection: using foreign aid shear activity report plasmid build kit to shear gRNA/Cas9,eventually making exogenous shear plasmid of luciferase coding regions can be translated into protein,then using fluorescein protein expression of the quantity to reflect the CRISPR/Cas9 effect.The plasmid transfection gRNA-Cas9 was performed in the nasopharyngeal carcinoma CNE-2 cells according to Lipofectamine2000 liposome transfection kit.The plasmid was extracted,and the CNE-2 cells were used to express the gRNA-Cas9 gene by using plasmid transfection technology to encode the gRNA-Cas9 gene sequence.Rt-pcr detected the content of EBNA1 and LMP1-specific primers,and EBNA1 and LMP 1-specific primers were amplified by the following steps.The expressions of EBNA1 and LMP1 were determined by Western blot method.Virus growth curve determination: the CRISPR/Cas9 EBNA1 and shear of EBV LMP1 and unused blank CRISPR/Cas9 shear EBV vaccination in platelet petri dishes,detect the virus drops degree change.Res?lts:CRISPR/Cas9 in targeted cut EBNA1 upstream of EBV gene sequences,the downstream target cut LMP1 sequence,gRNAs specificity of the sequence as indicated by the arrows(EBNA1-f,EBNA1-r,LMP1-f and LMP1-R)shear to the target site and binding sites,nasopharyngeal carcinoma CNE-2 cell transfection CRISPR/Cas9,over time,the stronger the luciferase,namely the longer duration of transfection,CRISPR/Cas9 CNE-2 nasopharyngeal carcinoma(NPC)cells to specific loci shear degree is higher,the latter into time dependence.In the case of CRISPR/Cas9 mediated,g RNA-Cas9 transfection with CNE-2 cells of the nasopharyngeal carcinoma(CNE-2 cells)was detected by Rt-PCR amplification of EBNA1-and lmp1-specific DNA strands in 48 hours after the amplification of the Rt-pcr,and the length of the DNA chain was equivalent to that of the unedited DNA chain.CRISPR/Cas9 technical editor EBNA1 and LMP1 in EBV,and edited the EBV infection to CNE-2 nasopharyngeal carcinoma(NPC)cells,the use of qRT-PCR measuring EBNA1 and LMP1 inhibition of nasopharyngeal carcinoma CNE-2 cells EBNA1 and LMP1 mRNA changes significantly decreased,the difference is statistically significant(p<0.01).CRISPR/Cas9 technical edi tor EBNA1 and LMP1 in EBV,and edited the EBV infection to CNE-2 nasopharyngeal carcinoma(NPC)cells,using Western blot method to measure LMP1 inhibition of nasopharyngeal carcinoma CNE-2 cells EBNA1 and LMP1 changes significantly decreased,the difference is statistically significant(p<0.01).Nasopharyngeal carcinoma CNE-2 cells infected with the extension of time,the virus drop degree increasing,by comparison,the difference is statistically significant.Western blot showed that the expression of Bax and Caspase3 were increased and the expression of Bcl-2 were decreased,the difference is statistically significant(p<0.01).Conclusion:EBV was edited by CRISPR/Cas9 technology in EBNA1 and LMP1,and the changes of EBNA1 and LMP1 mRNA and proteins of the nasopharyngeal carcinoma CNE-2 cells inhibited by EBNA1 and LMP1 were significantly decreased in the CNE-2 cells of nasopharyngeal carcinoma.And the growth of EBV was inhibited after edited.Moreover,the apoptisis of CNE-2 infected with this modified EBV was increased.