Effects Of RNA Interference Silencing POLD1 Gene On Cell Cycle-related Proteins

DNA polymerase delta,which plays an important role in DNA replication and damage repairation,is one of the main replicases in the process of eukaryotic DNA replication.p125,encoded by POLD1 gene,is the catalytic subunit of DNA polymerase delta and has poly erase and exonuclease activity.Researchs showed that abnormal expression of POLD1 gene had a close relationship with tumor occurrence and development,but its relationship with cell cycle-related proteins remained unclear.This research adopted the small RNA interference technology to knockdown POLD1 gene expression in HeLa,HUVEC and Chang Liver cells and tested the influence on expression level and distribution of cell cycle-related proteins by POLD1-siRNA.Methods:Cell morphology was detected by inverted microscope after POLD1-siRNA transfection;cell viability was by MTT;DNA damage by single cell gel electrophoresis and EB dying;the expression level and distribution of cyclins,CDK,CDKI,four DNA polymerase submits:p125,p50,p68 and p12 by immunofluorescence;expression level by p125 and P-actin.Result:The inhibition ratio of HeLa,HUVEC and Chang Liver cells after transfection 72 h were 15.75%,11.58%(p<0.05)and 22.49%(p<0.01)respectively,compared with control group.The inhibition ratio of HeLa and Chang Liver cell after transfection 72 h were 21.98%,and 13.65%(p<0.01)respectively,interestingly there was no significant difference on HUVEC cell vitality(p>0.05).After POLD1-siRNA1 transfected HeLa,HUVEC and Chang Liver cells,we found that cell number decreased,cell gap increased and cells turned slim.However cells turned flat and some bubbles appeared on the surface after POLD1-siRNA2 transfection.DNA damage were not detected,while early apoptosis were induced after POLD1siRNA transfection.After POLD1-siRNA transfected HeLa,HUVEC and Chang Liver cells,we found that CyclinD1,CyclinA2 and CyclinB1 proteins decreased.CyclinD1 protein was mainly distributed in the nucleus,CyclinA2 proteins was mainly distributed in the cytoplasm,CyclinB1 protein evenly distributed in the nucleus and cytoplasm.Low expression of CyclinD1,CyclinA2 and CyclinB1 protein can block cell cycle,thereby inhibiting cell proliferation.The impact of POLD1-siRNA on the expression and distribution of CDKs in POLD1-siRNA transfected HeLa,HUVEC and Chang Liver cells.CDK2 protein raised,while CDK4 protein decreased,CDK6 protein in HeLa cell decreased,while CDK6 protein in HUVEC cell raised.POLD1-siRNA1 induced CDK6 protein decreased and POLD1-siRNA2 induced CDK6 protein raised in Chang Liver cell,which is caused by sequence differences.CDK4 protein and CDK6 protein were mainly distributed in the nucleus.CDK4/CDK6 protein and CyclinDl protein were binding into complexes for cell cycle regulation,and CDK2 protein and CyclinE protein were binding into complexes for cell cycle regulation.Downregulation of CyclinD1 and CyclinE protein can block cell cycle,thereby inhibiting cell proliferation.The impact on the expression and distribution of CDKIs after POLD1-siRNA transfected HeLa,HUVEC and Chang Liver cells.p53 protein raised,p21 protein in HeLa and HUVEC cells raised,while p21 protein in Chang Liver cell decreased.p27 protein in HeLa and HUVEC cells decreased,while p27 protein did not change significantly in Chang Liver cell.p21,p27 and p53 proteins are mainly located in the nucleus.p21 and p53 can arrest the cell cycle regulation,thereby inhibiting cell proliferation.The impact on the expression and distribution of Pol? after POLD1-siRNA transfected HeLa,HUVEC and Chang Liver cells.p125 and p50 subunits were down.However,p68 and p12 subunits raised.p125,p50,p68 and p12 subunits are mainly distributed in the nucleus.p50 subunit p125 subunit is mutually binding,the binding is through the p68 subunit to solid.Conclusion:After POLD1-siRNA transfected HeLa,HUVEC and Chang Liver cells,we found that cell viability can significantly reduce and cell proliferation can be inhibited.Cells morphological changes can be induced by POLD1-siRNA.Cells slender types were induced by POLD1-siRNA1 and cells irregular were induced by POLD1-siRNA2.POLD1-siRNA transfected three cells,DNA does not receive damage,but nuclear morphology changes,which is induced early apoptotic cells.Expression of p125,CyclinD1,CDK4,CyclinA2 and CyclinB1 in HeLa,HUVEC and Chang Liver cells decreased,while CDK2 and p53 increased after POLD1-siRNA transfected,which can arrest the cell cycle regulation,thereby inhibiting cell proliferation.After POLD1-siRNA transfected HeLa,HUVEC and Chang Liver cells,we found that p125 and p50 subunits were down.However,p68 and p12 subunits are regulated.p125,p50,p68 and p12 subunits are mainly distributed in the nucleus.p50 subunit p125 subunit is mutually binding,the binding is through the p68 subunit to solid.After POLD1-siRNA transfection 72h in HeLa,HUVEC and Chang Liver cells,Cells were arrested in S phase,cell viability was inhibited,morphological changed,no DNA damage was detected,but the nuclear morphology changed,and early apoptosis happened.