Study On The Cannabidiol Induced Necroptosis And Optimization Of CRISPRi Efficiency

Cannabidiol(CBD)is one of the main cannabinoids of Cannabis sativa L.,has anti-tumor activity,and few side effects,it has been approved for adjuvant chemotherapy in Canada.We studied the effect of CBD on leukemia cell line K562 and found that CBD causes cell death with major vesicles at various doses,and chose 50 uM CBD for further study.To discern CBD caused which forms of programmed cell death,we observed the effects of small molecule inhibitors Necrostatin-1 and Z-VAD-FMK on CBD killing leukemia cells,and found that the inhibitor of RIPK1,Necrostatin-1,has a greater effect than Z-VAD-FMK,the Caspase inhibitor,suggests that CBD induces necroptosis in K562 cells.The endogenous ROS levels showed the first increase then decrease during CBD-induced cell death.We observed that the SOD mimetic tempol can delay the killing effect of CBD in K562,HL-60,and CEM/C1,after the treatment 6 h with CBD,the dead cell ratio among the three leukemia cell lines follows the pattern of K562>CEM/C1>HL-60.We quantitatively detected the endogenous ROS levels of three leukemia cells with CM-H2DCFDA dye and found that K562>HL-60>CEM/C1.Cells were then treated with TBHP to quantify the endogenous ROS level after oxidative stress and found that it followed the pattern of K562>CEM/C1>HL-60,which is the same as the programmed cell death ratio tent,suggesting that programmed cell death after CBD treatment is inversely proportional to cells ability to maintain the redox homeostasis.Transcriptome studies have shown that CBD activated the TNFa pathway,which may be the main pathway to activate necroptosis.The finding that CBD activated the TNFa pathway and increased endogenous ROS,to cause necroptosis of leukemia cells,the mechanism needs us to further study for laying the foundation for further development of CBD as a new anti-leukemia medicine.Knocking down the expression level of a gene is a common method for studying gene function,and we set out to determine how KRAB-dCas9 protein and sgRNA expression level affects the knockdown efficiency of CRISPRi system.K562 cell clones expressing KRAB-dCas9 protein either with the constitutive SFFV promotor or with the inducible Tet-on system were created by lenti viral transduction and fluorescence-activated cell sorting(FACS).The sgRNA of six genes from two libraries was used to test the knockdown efficiency of the CRISPRi system in four cell clones.We determined the expression level of the KRAB-dCas9 protein/sgRNA level by flow cytometry.The result showed the highest KRAB-dCas9 expression level is the precondition of the effective knockdown efficiency of the CRISPRi system,74.72%,72.28%,39.08%knockdown efficiency of mmadhc,rpia,znf148 genes were achieved.Subsequently,we modified the expression level of sgRNA by adopting different multiplicity of infection(MOI)in lentiviral transduction and found that the knockdown efficiency correlated with sgRNA expression level.Linear regression modeling of the knockdown efficiency data revealed that the sgRNA level has a greater impact than the expression level of KRAB-dCas9 protein for the knockdown efficiency of the CRISPRi system.The result indicating that the sgRNA level is a major factor affecting CRISPRi efficiency based on the cell clone with enough KRAB-dCas9 protein expression.