Effect Of HDAC2 Sumo-E3 Ligase Mutant On The Proliferation And Migration Of DLD1 Cells

Objective:To investigate the effect of HDAC2 sumo-E3 ligase segment on proliferation and migration of DLD1 cells and the potential mechanisms.Methods:The whole gene synthesis obtained HDAC2 sumo-E3 ligase functional domain fragment HDAC2325-488,that is,from the 325th amino acid to the 488th carboxyl-terminal corresponding coding gene sequence.And to construct its recombinant lentiviral expression vector.Prepare 5×PEG-8000+NaCl（weigh NaCl 8.766 g,PEG8000 50 g,dissolved in 200ml Milli-Q pure water）,sterilized at 121℃for 30 min,and store at 4℃.The lentiviral supernatant was filtered using a 0.45μm filter head.Each 30 ml of the filtered virus initial solution was added with 7.5 ml of 5×PEG-8000+NaCl mother liquor.The mixture was mixed every 20-30 min for a total of 3-5 times.4℃overnight;4℃,4000 g,centrifugation for 20 min;aspirate the supernatant,stand the tube for 1-2 min,and aspirate the residual liquid;add the appropriate amount of lentivirus solution to dissolve the lentivirus;Lentivirus after collection The suspension was divided into 50μl portions and stored in the finished tube.Store in a refrigerator at-80℃.The DLD1^hdac2-/-cells were plated into 6-well plates at a density of 30%to 50%.After l8h lentivirus infection for 48 hours,the cells were digested and then serially diluted to 10 cells/ml in 10-fold increments to inoculate 96-well plates at100μl./Well,1 cell per well,single cell clones were observed after 7 days of culture,and positive clones expressing green fluorescent protein were selected to continue the expansion culture.At the same time,the photos were taken with no-load pairs and GFP-labeled DLD1^hdac2-/-cells were obtained.Western blot was used to further identify the DLD1h325-488 cell line expressing exogenous HDAC2（325-488）.The well-developed human embryonic kidney 293FT cells were inoculated into a 10cm dish at a density of 50%to 70%,and were used for transfection after the cells were adherent for 24 hours,and infected DLD1^hdac2-/.-Cells（without endogenous HDAC2 expression）,screen cell clones DLD1^h325-488325-488 stabilizing HDAC2（325-488）;prepare 5x PEG-8000+Na Cl for virus collection and concentration.The proliferation and migration of DLD1^h325-488325-488 stable cells were detected by RTCA real-time cell analyzer,Trans-well,etc.The expression of DLD1^h325-488325-488 stable cell proliferation and migration-related genes was detected by immunoblotting and qRT-PCR.Results:The DLD1^h325-488325-488 cell line stably expressing the HDAC2 sumo-E3 ligase mutant HDAC2（325-488）was constructed,and DLD1^h325-488325-488 cells stably expressing the HDAC2sumo-E3 ligase mutant HDAC2（325-488）were obtained.DLD1^hdac2-/-cells were infected with lentivirus for 48h.The growth of cells was observed under fluorescence microscope.Green fluorescence was observed.The FLAG-tag antibody assay identified a single cell clone that expresses exogenous HDAC2（325-488）.DLD1^hadc2-/-and DLD1^h325-488325-488 cells were digested and prepared with serum-free DMEM medium to make a cell suspension of 1×10⁶cells/ml.500μl of 10%FBS DMEM medium was added to a 24-well plate.The Tran-swell chamber（Millipore,membrane with a pore size of 8μm）was placed on the well plate,and100μl of cell suspension per well was added to each well.1 x 10⁵cells,3 replicates in each group.24 hours after incubation in the cell incubator,the chamber was removed,ethanol was fixed for 15 minutes,and 0.1%crystal violet was used for staining for 10 minutes.The cells were gently wiped off with a cotton swab.The cells were photographed under a microscope to observe the number of cells on the lower surface of the chamber to determine the cells migration situation.RTCA real-time cell analysis assays further confirmed that the expression of HDAC2（325-488）slightly promoted the cell proliferation index,but had no significant effect.This suggests that HDAC2 sumo-E3 ligase activity plays a minor role in the proliferation of DLD1 cells.Western blot results showed that the expression of MMP14protein in DLD1^h325-488325-488 cells was significantly up-regulated compared with DLD1^hdac2-/-cells,suggesting that HDAC2 sumo-E3 ligase regulates post-transcriptional expression of MMP14.On the other hand,compared with DLD1^hdac2-/-cells,the expression levels of cyclinD1,ODC1 and other proteins in DLD ^h325-488325-488 cells were not significantly up-regulated,which was consistent with no significant changes in cell proliferation activity.Conclusion:HDAC2 sumo-E3 ligase segment may promote the migration of DLD1 cells by up regulating MMP14 expression.