PARP1 In ?-lapachone Destruction Effect And Mechanism In Gastric Cancer Cells

Objective:To investigate the role and molecular mechanism of PARP1 in the killing of human AGS gastric cancer cell lines by ?-lapachone.Method:1.Human AGS gastric cancer cell lines was used as the research object,and 3 siRNA oligonucleotide gene fragments were designed and synthesized for the target gene PARP1,as well as the negative control group(NC-siRNA).The interference of PARP-1 expression with the method of conventional transient transfection.2.After transfection of 4 groups were added 4umol/1 ?-lapachone,Western blot assay was used to detect the expression changes of PARP1 cleavage product protein in four groups,Variance analysis was used to compare,Stable transfected cell lines were selected.3.The screened siRNA was selected for cell transfection,and the negative control group NC-siRNA transfection group was set up.The experiment of MTT and Plate cloning experiment were detected the gene transfection group(PARP1-siRNA group),NC-siRNA transfection group of ?-lapachone on the proliferation and clone ability of AGS gastric cancer cell lines.The Scratch experiment were detected the gene transfection group(PARP1-siRNA group),NC-siRNA transfection group of P-lapachone on the migration ability of AGS gastric cancer cell lines.Results:1.Transfection efficiency of AGS gastric cancer cell lines after transfection:Cells transfected 48h cells were observed under the microscope,compared with the NC-siRNA group,PARP1 group of gene silencing in morphology did not change significantly,round,adherent growth,PARP1 gene silencing had no significant effect on cell morphology.Western blot assay(joined 4umol/1?-lapachone)was used to detect the expression changes of PARP 1 gene product protein 72h after transfection;Compared with NC-siRNA,the expression of PARP 1 cleavage product protein decreased significantly after transfection,PARP1-siRNA 1 decreased by 89%(p<0.01),PARP 1-siRNA 2 decreased by 73%(p<0.05),and PARPl-siRNA 3 decreased by 83%(p<0.05),Compared with the other three groups,the expression level of PARP 1-siRNA 1 protein in group was lowest after transfection,indicating that PARP1-siRNA 1 is the most specific and effective sequence,which canbe used to interfere with the deletion of PARP1 gene,and is used for subsequent experiments.2.Effects of PARP1-siRNA transfection on proliferation,clone and migration of AGS gastric cancer cell lines:MTT,Plate cloning experiment,Scratch experiment can be observed with ?-lapachone could significantly inhibit the NC-siRNA transfection group cells proliferation,cloning and migration,but no obvious inhibitory effect on PARP1-siRNA transfection group.siRNA interference of PARP1 gene expression can significantly inhibit the cell destruction caused by p-lapachone.Conclusion:I.The use of RNA interference technology can successfully inhibit the expression of PARP 1 gene in AGS gastric cancer cell lines.2.Excessive activation of PARP1 plays a key role in the killing of gastric cancer cells by?-lapachone.