The Effects Of P300 HAT Domain Mutation On The Biological Function Of Gastric Cancer Cell BGC-823 And Its Mechanism Of Proliferative Inhibition

Background and ObjectiveGastric cancer is one of the most common cancers in the world.Its fatality rate accounts for about 10% of cancer deaths and ranks third among cancer-related deaths.It is concealed in the early stage and not easy to be detected.Surgical treatment is often used,and the recurrence rate is high.Most of them were late when they were diagnosed.For advanced gastric cancer,chemotherapy is the main treatment method,and its median survival time is less than 1 year.Therefore,it is urgent to explore new targets for the treatment of gastric cancer.The transcription cofactor p300 plays a role in a variety of functions.It possesses histone acetyltransferase activity,HAT domain plays a role in acetylation of p300.Some studies have shown that small molecule inhibitors acting on the HAT domain inhibit the proliferation of tumor cells after inhibiting the function of p300/CBP.In gastric cancer tissues,the expression of p300 is abnormal.There are few studies about p300 HAT domain in gastric cancer,and there are no report focusing on the knockout of the p300 HAT domain.Therefore,we used CRISPR/Cas9 technology to construct gastric cancer cell line with the p300 HAT domain mutation,and research its effect on the proliferation of gastric cancer cells and explore its underlying mechanism.Methods1.px459-HAT-sgRNA recombinant vectors were ConstructedUsing the online website http://crispr.mit.edu/,we designed the sgRNA sequence for the fifth exon of p300 HAT domain,after synthesis,ligated to the px459 vector to construct the px459-HAT-sgRNA recombinant vectors,transfected them into human embryo kidney HEK293 T in advance to verify their cleavage activity.2.Monoclonal gastric cancer cell line with mutation in the HAT domain of p300 was establishedGastric cancer cells BGC-823 were transfected with the px459-HAT-sgRNA recombinant vectors by cell transfection technology and screened with puromycin.The limiting dilution method was used to select and culture single clones.Extract the genome and magnify DNA target fragment by PCR,pick out the PCR products with homozygous deletion by agarose electrophoresis and send them for sequencing,to determine whether the HAT domain of p300 is successfully knocked out.Western blot was used to identify the protein.3.To detect the effect of mutation the HAT domain of p300 on the biological function of cellsUnder the microscope,we observed the changes in cell morphology.Cell proliferation ability was detected by MTT experiment.Scratch test was used to detect cell migration ability,transwell experiment was used to detect cell invasion ability,and flow cytometry was used to detect the cell cycle.4.Detection of differences in gene expression after mutation in the p300 HAT domainRNA-seq was used to detect differential genes between the p300 HAT Mut group and the control group.5.To verify differential genes from mRNA level and protein levelqPCR and Western Blot technology were used to verify from mRNA level and protein level respectively.Result1.The px459-HAT-sgRNA recombinant vector was successfully constructed.2.A monoclonal gastric cancer cell line with mutation of p300 HAT domain was successfully obtained.The fragment of 66 bp was missing,which met our expectations.3.Cell morphology changes after mutation the HAT domain of p300,the cell volume becomed smaller and rounder.Proliferation ability of the cell reduced,the migration and invasion ability is also inhibited,and the cell cycle was blocked.4.There are 2442 differential genes found in HAT Mut cells group compared with control cells,of which 1497 genes were up-regulated and 945 genes were down-regulated.We selected p21 and cyclin D1 from the down-regulated genes to study the underlying mechanism of inducing changes of cell biological function.5.p21 and cyclin D1 at mRNA and protein levels were down-regulated.ConclusionsMigration and invasion of gastric cancer cell BGC-823 was inhibited,and after mutation of p300 HAT domain;cell proliferation was inhibited and cell cycle was blocked,which may be related to down-regulation of p21 and cyclin D1 gene expression by the change of p300 HAT structure.