Research On Overexpression Of Separase By Tet-on System Regulating And Effect On Biological Character In Hela Cells

BackgroundSeparase resolves cohesin/SCC1 which results in separating of sister chromatid. It is perfect space-time regulation event that sister chromatid separates. If separating of sister chromatid is disorder, genetic material instability and corpuscular aneuploidy will appear, that can lead to be fatality of cell. The chromosome is the elementary structure that ensures genetic materials transmit shabbily, integraly and correctly. The two sister chromatids connect each other only at centromere during the mitosis metaphase and the main structural protein of centromere--cohesin is degraded during the mitosis anaphase. The two sister chromatids are segregated by spindle finber traction and allocted to two daughter cells. Now generally consider a conservative mechanism participate separating of sister chromatid that is acknowledged, following with completion of sister chromatid duplicating, sister chromatid is connected by cohesion. In mitosis anaphase, securin is depredated by ubiquitin of APC/C, thereby separase is activated and split cohesion,and then sister chromatid separate. In vertebrate, decollement of cohesin include two steps: first step, in prophase and premetaphase, the cohesin of chromosomal arm is splited, Pole-like kinase(Plk1) and Aurora-B is essential, especially, Plk1 make scc3 and SA2 phosphorylation which play a important role. The second step, in anaphase, the cohesin of centriolar region is splited by securing-separase path. Shugoshins protect the cohesin of centriolar region which avoid to be decomposed in prophase.Abnomal expression of separase can influence genome instability and tumor sensitivity. Waizenegger etc discover, in homo-sapiens cells, after expression of separase is refrained by RNAi, there appear polyploid cells with large lobiform nuclear and sister chromatid of nondisjunction. Karin G. Wirth etc have an experiment, knockout alleles of the mouse Separase, blocks sister chromatid separation but does not prevent other aspects of mitosis, cytokinesis, or chromosome replication.Separase depletion from human cells using RNA interference. Hela cells lacking separase are delayed or arrest at the G2-M phase (Cell Cycle. 2005 Nov. 4(11):1576-84). The securin-separase complex might aid DNA repair by removing local cohesin in interphase cells. Securin is essential for separase stability and also for proper repair of DNA damaged by ultraviolet, X-ray and gamma-ray irradiation.(Nature. 2004 Aug 26.430(7003):1044-8. ). Jennifer L. Shepard etc have a experiment on zebra, mutagen affect zebra of separase gene mutation which lead to increase 2.5 fold of all tumor and 8 fold of endepidermis tumor.Separase can promote cell apoptosis. Uhlmann etc pointed out that Esp1(separase)is similar to caspases. The two belong to cysteine proteinase, contain conservative Cysteine residue which is catalytic activity group of caspases. Pati and Chen introduced: spallation of cohesion/ Rad21 subunit cause apoptosis. In 2005, Court reported: expression of Rad21 was decreased by RNAi, and then tumor cell proliferation was refrained, apoptosis added. Hui Yang etc had a finding: separase can cut mcd1 and induce apoptosis by hydrogen dioxide induction.Separase have an important role in cell cycle, genetic material transmission, genome instability, tumor sensitivity, apoptosis. Now that, there are some findings that separase of low or negative expression induce tumor, we plan to make separase of overexpression and observe biological character change. Why do we select Hela cells as research subject? Main cause is that Hela cells chromosomes (85) have a obviously difference with cervix uteri epithelial cell ones (46). Following our study progress, we confirm that separase of overexpression can deteriorate malignant of Hela cells, whether these are concerned with Hela cells apoptosis. We have a research on expression of separase by Tet-on system regulating and its effect on biological character in Hela cells.Methods and results1.Tet-on expressing control model was constructedPsk-Esp1 plasmid was cut by EcoRâ… and Sacâ…¡, objective fragment Esp1 was retrieved by gum, and then inserted pTRE-d2EGFP plasmid, transfected Bacillus coli, selected correct recons which were identified inserting direction by Xhoâ… and Sphâ… , Sequencing identified, Esp1-pTRE-d2EGFP plasmid was constructed. PTet-on plasmid was transfected into Hela cells by FuGENE 6 liposome, G418 of 200ng/ ml screen after 24 hours, monoclone was picked after 2 weeks. Esp1-pTRE-d2EGFP plasmid and PTK-Hyg plasmid according to molar ratio 20:1 were transfected into Hela cells with PTet-on plasmid, G418 of 600ng/ ml and homomycin of 200ng/ ml screen after 24 hours, monoclone was picked after 2 weeks.expression of Esp1 gene was detected by real time PCR and WB in mRNA and protein level. We draw a result: optimal induction concentration of Doxycycline is 200ng/ ml, expression of Esp1 gene is augmentation in mRNA and protein level.2.Overexpression of Esp1 gene effect on ploidy in Hela cells:We collected the Hela cells induced by optimization concentration dox and dealed with colchicine, produced chromosome, and then counted chromosome by artificial and VIDEOTEST-KARYO apparatus, analyzed DNA ploidy by flow cytometry. We draw a result: after overexpression of Esp1 gene, chromosome mean of Hela cells droped from 83 strips to 71 strips, DI of Hela cells droped from 2.2 to 1.89.3.Overexpression of Esp1 gene effect on apoptosis in Hela cells:With optimization concentration dox, we induced apoptosis in Hela cells by Etoposide(VP-16)and X-ray, detected with FITC-Annexin V in flow cytometry. We draw a result: after overexpression of Esp1 gene, apoptosis rate of Hela cells increase from 2.3% to 19.4% by Etoposide and from 3.8% to 14.8% by X-ray.Conclusion1.Tet-on expressing control model was constructed which conduce to observe the effect on Hela cells of Esp1 gene expression from low to high density.following density of DOX increasing, the expression of Esp1 added in timing. Following Tet-on system constructed, we can observe overexpression of separase and the effect on character in Hela cells, control expression level of separase. Next way, we will study gene function and gene therapy.2.Overexpression of Esp1 gene lead to decrease chromosome number of Hela cells, degrade DNA ploidy. According records, separase and cohensin subunit Rad21 not only can repair impaired DNA, also promote chromosome correct separation. All over, overexpression separase can promote tumor cells chromosome separation and reverse Hela cells malignant degree.3.Overexpression of Esp1 gene increase apoptosis rate of Hela cells by X-ray and VP-16. Two different apoptosis path induction can increase apoptosis, which cue to that separase contribution in apoptosis is the posterior of X-ray and VP-16 apoptosis path crossover.In a word, a controlling and inducing Tet-on system model constructed in our experiment, and then draw a conclusion that chromosome number of Hela cells is lower in some level and apoptosis rate increase with overexpression of Esp1 gene.