Cell Morphological Change And Function Abnormality In HepG2 And 293T Cells With LMNA Knockout

Chapter 1.Knockout of human LMNA gene by CRISPR/Cas9 system in HepG2 and 293 T CellsObjective: To edit the LMNA gene of 293 T and HepG2 cell lines cultured in vitro using CRISPR/Cas9 technology to obtain LMNA knockout cell lines.And observe the changes in cell morphology and cell morphology.Methods: The LMNA gene of 293 T and HepG2 cells was edited by CRISPR/Cas9 system,and the stable cell lines were obtained by puromycin selection and monoclonal selection.And verified by sequencing and western blot.The surface morphology of the cell membrane was observed by scanning electron microscopy.The nuclear membrane morphology was observed by transmission electron microscopy.The nuclear membrane and cytoskeleton were observed by immunofluorescence technique.Results: Both 293 T and HepG2 cells had base deletions.Western blotting showed that Lamin A/C was not expressed,and two LMNA knockout cells(LMNA KO)were successfully obtained.After LMNA knockout,the 293 T cell body became smaller and rounded,and the prominent junction was reduced and thinned;the HepG2 wild type was long spindle type,the nucleus was prominent in the middle,and the cytoplasm was mainly distributed at the both ends of the cell type,Lamin A/C knocking.After the removal,the cells become polygonal,and the cytoplasm is more evenly distributed in the perinuclear.Transmission electron microscopy revealed that after the LMNA gene was knocked out,the cell membrane became uneven.Conclusion: The deletion of Lamin A/C leads to redistribution of the cytoskeleton,which in turn changes cell morphology.Chapter 2.Cell Function Abnormality In HepG2 and 293 T Cells With LMNA KnockoutObjective: To investigate the effects of LMNA gene on the tumorigenicity of tumor cells by in vivo and in vitro tumorigenicity experiments on 293 T and HepG2 LMNA KO and wild type(WT),and to analyze the related molecular mechanisms.Methods: 1)293T and HepG2 cells were divided into wild type group and LMNA KO group.The proliferation of four cells was detected by CCK-8 method.The apoptosis and cell cycle of four cells were detected by flow cytometry.LMNA was overexpressed,and the expression levels of cell cycle-associated protein CDK1 and Cyclin B1,DNA damage-related protein ?-H2 A.X and tumor suppressor gene P16 were detected by western blot,and the expression levels of histone H3K9me3,H3K27me3 and RB.2)Detection of migration and perforation ability of wild-type and LMNA KO cells by cell scratches and transwell.The expression levels of MMP2,MMP9,COL1A1 and integrin ?4 were detected by western blot.3)Soft agar colony formation experiment,3000 cells were added to each well of 6-well plate,and the diameter and number of clones were measured after 4 weeks;plate clone formation experiment,1000 cells were added to each well of six-well plate,and clones were counted 2 weeks later.Quantity.Twenty-four 4-week-old nude mice were randomly divided into 4 groups,and wild type and LMNA KO type cells of 293 T and HepG2 were injected respectively.Each nude mouse was injected with 2×10/6 cells on both sides of the forelimb,and two per week were measured.The diameter of the secondary tumor,the mice were sacrificed by cervical dislocation after 3 weeks,and the tumor mass was measured to measure the volume and weight.Results: 1)The proliferation of 293T-LMNA-KO was lower than that of 293T-WT(P<0.05),the apoptosis rate increased(P<0.05),and the G2/M phase arrest,Cyclin B1,CDK4/6,H3K27me3 H3K9me3 did not change significantly,CDK1 decreased(P<0.05),? H2 A.X expression increased(P<0.05),P16 expression increased(P<0.05);HepG2-LMNA-KO significantly decreased compared with HepG2-WT(P<0.01),the apoptosis rate increased(P<0.05),and G2/M phase arrest occurred;Cyclin B1,CDK4/6,H3K27me3,H3K9me3 did not change significantly,CDK1 decreased(P<0.05),? H2 A.X The expression increased(P<0.05)and the expression of P16 increased(P<0.05).2)293T-LMNA-KO showed a significant decrease in horizontal migration ability(P<0.001),transwell perforation ability(P<0.05),increased MMP2 expression,and integrin ?4 activation;HepG2-LMNA-KO The level of migration ability of HepG2-WT cells was significantly decreased(P<0.001),the transwell ability of transwell was enhanced(P<0.01),the expression of MMP2 was increased,and the expression of integrin ?4 was activated.3)The number of 293T-LMNA-KO clones decreased compared with 293T-WT soft agar(P<0.05),the diameter of clones decreased(P<0.05),the number of clones in plate clones decreased(P<0.05),and tumors were formed in nude mice.The volume and mass of the experimental tumors decreased(P<0.05).The number of HepG2-LMNA-KO clones decreased compared with HepG2-WT soft agar(P<0.05),the diameter of clones decreased(P<0.05),and the number of clones in plate clones.The decrease(P<0.05),the tumor volume of the tumor-forming tumors in nude mice decreased(P<0.05),and the quality decreased(P<0.01).Conclusion: The proliferation of the two LMNA KO cells decreased,the migration ability decreased,the perforation ability increased,the soft agar clone formation ability decreased,there was G2/M phase arrest,and the tumorigenic ability in nude mice also decreased significantly.Later,it was found that knocking out the LMNA gene in tumor cells,the expression of P16 increased,and the expression of CDK1 decreased,leading to cell cycle arrest,which ultimately caused the tumorigenic ability of tumor cells to decrease.