Effects Of Rna Interference Silencing Pold1Gene On Cytoskeleton And Cell Migration

DNA polymerase delta (Pold), one of the key replication enzymes in eukaryotic cells, plays a central role in DNA replication and genome maintenance. Pold is encoded by POLDl gene, and the p125catalytic subunit of it has the polymerase and exonuclease activity. Studies have found that there was a direct relationship between the abnormal expression of POLD1gene and the occurrence and development of cancer. However, the precise mechanisms underlying down-regulation POLD1in tumorigenesis remain unclear. In this study, we applie small interfering RNA to knockdown expression of POLD1gene in HeLa, HUVEC and Chang Liver cells.Then these effects to the distribution of cytoskeleton are detected. We want to find the correlation of knockdown POLD1with cytoskeleton and cell migration in HeLa, HUVEC and Chang Liver.Methods:1. Design and synthesize the siRNAs that contrapose POLD1gene of homo sapiens, and screening out the effective POLD1-siRNAs (POLD1,2). Cells in logarithmic growth phase were used in this study, and the effect of POLD1-siRNA on cell vitality was detected by MTT. Cell migration was tested by wound healing in HeLa, HUVEC and Chang Liver.2. After the cells were transfected with differetnt POLDl-siRNA for72h, the cell morphology changes were observed by inverted microscope.3. Confocal microscopy observed the distribution and content of microtubule, microfilament and intermediate filaments, for example, a-Tubulin, y-Tubulin, F-actin, Cortactin, FilaminA, Vimentin and LaminB.4. The distribution and content of Paxillin, FAK and Src was detected by immunofluorescence.5. Immunofluorescence analysis and confocal microscopy observe the distribution and content of p125, Rho and Cdc42. The expression of p125was detected by Western blotting.Results:1. After HeLa, HUVEC and Chang Liver cells were transfected with POLDl-siRNAl for72h, the inhibition rate of HeLa and HUVEC were11.38%and12.11%respectively. Compared with the control group, cell vitality was decreased significantly (p<0.05). The rate of Chang Liver was21.32%(p<.01).After HeLa, HUVEC and Chang Liver cells were transfected with POLD1-siRNA2for72h, the inhibition rate to HeLa were17.85%. Compared with the control group, cell vitality was decreased significantly (p<0.01). However, the rate of HUVEC and Chang Liver was no significant difference (p>0.05).2. The results of wound healing test showed that the wound healing rate of HeLa was inhibited before48h. After48h inhibition is abate and had a tendency to promote the migration after treatment with POLD1-siRNA. The ability of migration was increasing obviously in HUVEC. But the wound healing rate of Chang Liver was lower, and the ability migration decrease.3. After HeLa and HUVEC cells were transfected with POLDl-siRNAl for72h, the cell morphology turn to long and thin. Microtubules were bunchy and distributed asymmetrically. In the front, microtubule structure were stabled and the number increases, it decided the direction of cell migration. Microfilament gathered in the cell membrane, formed filopodia and lamellipodium. In the end, Microfilament participated in the process of cell migration. Vimentin was a sign of cell migration. The content of vimentin increased and promoted the migration of HeLa, HUVEC cells. Howerver, after Chang Liver cells were transfected with POLD1-siRNA1for72h, Microtubule organization for dense mesh structure and microfilament depolymerized. The vimentin was down-regulated. These data indicated knockdown PODLl-siRNAl markedly inhibit the migration of Chang Liver cells.After HeLa and HUVEC cells were transfected with POLD1-siRNA2for72h, the cell morphology changed irregular. Filopodia and lamellipodium were formed in cytomembrane. In cell front, microtubule and microfilament polymerized in order to induce cell migration. Microfilament gathered in the cell membrane. But in Chang Liver cells, microfilament depolymerized. At the same time, the vimentin was down-regulated. So the ability of Chang Liver cell migration was reduced.4. After HeLa and HUVEC cells were transfected with POLD1-siRNAl for72h, the expression of FAK and Src was increased and the FAK/Src kinase was activated. And then the sscaffold protein paxillin was phosphorylated by the FAK/Src to induce cell migration. Meanwhile, the distribution of HeLa’s FAK in nucleus increased and regulated cell migration.After HeLa and HUVEC cells were transfected with POLD1-siRNA2for72h, the content of FAK and Src was increased obviously inducing cell migration. But the FAK was down-regulated in HUVEC. These data indicated FAK was of no effect on cell migration by transfected with POLD1-siRNA2in HUVEC.5. After HeLa and HUVEC cells were transfected with POLD1-siRNAl for72h, Rho in two cells was up-regulated, which associated with the form of stress fiber and Lamellipodium, resulting in a increase in cell migration. However, the expression of Rho decreased Chang Liver migration.After HeLa cells were transfected with POLD1-siRNA2for72h, the activation of Rho promoted cell migration. But the low expression of Rho may not be involved in HUVEC cell migration. The POLD1-siRNA knockdown in Chang Liver cell up-regulated Rho, resulting in a decrease in cell migration.Conclusions:The transfection of POLDl-siRNA1induced activation of FAK and Src, up-regulated the expression of FAK and Src. The activation of FAK and Src enabled Paxilliin to phosphorylate. At then HeLa and HUVEC cells front adhension ability turn to strong, the adhension between focal adhesion and extracellular matrix in cell edge disappear inducing cell migration.After treated with POLD1-siRNA2for72h, the activation of FAK-Src pathways promote cell migration in HeLa cell. However, the expression of FAK was decreased without activation in HUVEC cell.The transfection POLDl-siRNAl induced stress fiber polymerizing, filopodia and lamellipodium forming by Rho, resulting in increase in cell migration in HeLa and HUVEC cell.After treated with POLD-siRNA2for72h, the activation of Rho promoted cell migration in HeLa cell. However, the expression of Rho was decreased without activation in HUVEC cell.In conclusion, cell migration might be regulated by FAK-Src signaling and Rho GTPases.