The Effect Of PA28α On Proliferation, Apoptosis And Migration Of Esophageal Squamous Cancer Cells

Proteasome is a kind of many catalytic sites protease holocomplex, which isresponsible for the most of the protein degradation in cells. By regulating degradationof damaged or misfolded proteins, proteasome regulates the cellular process, such asthe cell cycle, DNA repair, apoptosis, signal transduction and immune response andso on. The most common form of proteasome is26S proteasome, which consists of20S core particles and proteasome activators. In the effect of proteasome activator,protein substrates turn into the20S core particles to be degraded.Esophageal squamous cell cancer (ESCC) is one of the malignant tumor in thenorth of China. ESCC is difficult to diagnose and easy to relapse, which lead to thelow5-year survival rate of patients. Current methods for the treatment of esophagealcancer are not optimistic. This situation prompted us to investigate the mechanism ofesophageal squamous cell carcinomas. In order to reduce the incidence of esophagealsquamous carcinoma, research and development of new drugs should be provided forthe treatment of esophageal cancer.At present, a number of studies have found that the abnormal phenomenon ofPA28expression in the malignant tumors of human. Zhang Lin found PA28α caninhibit the gastric cancer cell growth, proliferation and the cell cycle. Lemaire detected that PA28α had different location in ovarian epithelial cancer cells.Therefore, PA28α can be used as a potential biomarker of ovarian cancer. Researcheshave shown that the other proteasome subunits can be used as the target of tumortherapy. Studies have shown that PA28γ has a significant increasing in breast cancerwhich enhance the capacity of the cancer cell migration.In our work, we verified the RNA and protein levels of PA28α in the normalesophageal epithelial Het-1A cells, Eca109and EC9706. The effects of PA28αknockdown on the proliferation, apoptosis and migration of Eca109were also tested.This topic mainly divided into two parts:Part1: We compared the RNA and protein levels of PA28α in Het-1A, Eca109and EC9706. What’s more, we observed the location of PA28α in the cells under thefluorescence microscope.Part2: We tested the role of PA28α in occurrence and development ofesophageal squamous cancer cells. Treatment with siRNA for PA28α and IFN-γsignificantly affected the proliferation, apoptosis, migration of Eca109.Materials and Methods1. Cell linesThe normal esophageal epithelial Het-1A cells and the ESCC cell line EC9706cells, Eca109cells were all saved by our laboratory. All of cells in our work werecultured in RPMI1640medium supplemented with10%standard fetal bovine serum(FBS) at37℃, under saturated humidity,5%CO2incubator.2. The expression and location of PA28α in Het-1A and ECSScellsWe analyzed the expression and location of PA28α in Eca109, EC9706andHet-1A by Real-time quantitative PCR (qRT-PCR) and Western Blot. We also observed the location of PA28α protein in the cells.3. Effect of siRNA-mediated down-regulation of PA28α on theproliferation, apoptosis and migration of esophageal squamouscancer cells in vitroFirstly, we optimized the most effective interference fragment. Next, we used thebest interference fragment to screen the best interference fragment concentrations.And then we used effective interference fragment and the best concentration ofinterference fragment to control the expression of PA28α protein in esophageal cancercells. Lastly, we tested the effect of siRNA-mediated down-regulation of PA28α onthe proliferation by EdU and CCK-8assay, apoptosis by Annexin V-FITC/PI FlowCytometry and the expression of the activation of caspase-3, migration by transwelland the expression of E-cadherin.Results1. The expression and location of PA28α in the normalesophageal epithelial Het-1A and ECSSqRT-PCR and Western Blot results showed that, compared with Het-1A cells,PA28α showed higher expression in Eca109and EC9706cells, especially in Eca109cells. Cell immunofluorescence test showed: PA28α protein mainly locates incytoplasm of esophageal squamous cancer cells.2. Effect of siRNA-mediated down-regulation of PA28α on theproliferation, apoptosis and migration of esophageal squamouscancer cells Eca109in vitro Compared with control groups, siRNA-mediated down-regulation of PA28α caninhibit the proliferation of Eca109, to some extent, can enhance the apoptosis ofability. It may be due to that PA28α influences the proteasome activity leading to thechange of the proliferation and apoptosis ability of Eca109cells. The results alsoshowed that the proteasome plays a certain role in esophageal squamous carcinoma.What’s more, siRNA-mediated down-regulation of PA28α can inhibit the migrationof Eca109cells and the expression of E-cadherin.Conclusion1. Compared with the normal esophageal epithelial Het-1A cells, PA28α showshigher expression in Eca109and EC9706cells, especially in Eca109cells.2. PA28α protein mainly locates in cytoplasm of esophageal squamous cancer cells.3. Compared with control groups, siRNA-mediated down-regulation of PA28α caninhibits the proliferation of Eca109, enhance the apoptosis of ability, inhibit themigration of Eca109cells and the expression of E-cadherin.