The Roles Of Proteasome Subunit PSMD7in Ciliary Or Flagellar Disassembly And Carcinogenesis Of The Esophagus

The ubiquitin proteasome system (UPS) is the major protein degradation system in eukaryotic cells and regulates a variety of cellular functions, including removal of damaged proteins, cell cycle regulation, apoptosis and signal transduction. Cancer cells depend on high proteasome activity to remove harmful immature and irregular proteins generated by rapid proliferation and to strength the anti-apoptosis ability. Studies have shown that the ubiquitin-UNTjunction system are present in flagella of model organism of algae Chlamydomonas reinhardtii and Dunaliella salina, and recently it has also been identified in the ciliome of Caenorhabditis elegans, Drosophila melanogaster, Mus musculus, and Homo sapiens. So we infer that UPS may be involved in the biological function of cilia/flagella.Cilia and flagella are UNTserved organelles during evolution with similar structure and origin from basal body protruding from cell surface. Flagella exist in lower eukaryotes whose genome structures are more simple and easy to study. The intraflagellar transport mechanism was first identified in the model organisms Chlamydomonas reinhardtii, and then was UNTfirmed in the cilia of higher organelle. Nearly all kinds of human cells have cilia. Cilia disassembly is closely related to cell-cycle Control, which releases the centrosome to form the mitotic spindle and cilia are regenerated after the completion of cell cycle. Tumor cells usually have abnormal cilia and there are multiple tumor-related signaling pathways in cilia, but the molecular mechanism between them is not clear. A large number of ciliary proteins will be degraded during disassembly. Although the ubiquitin UNTjunction system has been identified in cilia/flagella, whether proteasome is involved in the ciliary/flagellar disassembly and whether UPS could be involved in the tumorigenesis remains unclear.However, cilia are hard to be observed and disappeared during cell culture in vitro. Flagella are longer and can be induce to disassembly by simple operation which could be used as model organelle to help us to better understand the molecular mechanism between UPS and ciliary disassembly. Dunaliella salina and Chlamydomonas reinhardtii are eukaryotic green algae with the difference that the former has a layer of cell wall but the latter not. The flagellar structure of Dunaliella salina is more similar with mammalian cilia. So the flagella of Dunaliella salina were used as a model organelle in this study to provide evidence for the research of cilia and tumorigenesis.Based on both structure and function the proteasome can be further divided into two sub-complexes, a central20S proteolytic core particle (CP) that is capped at either end by one or two19S regulatory particles (19S RP). The20S CP is the degradation unit and UNTtains the active sites enquired to hydrolyze proteins into peptides. The19S RP Controls the degradation of ubiquitin-tagged substrates by acting as a receptor for polyubiquitinated proteins and facilitating their ATP-dependent translocation into the catalytic chamber of the20S CP. Inhibitors of20S CP bortezomib is used for treatment of multiple myeloma and lymphoma, but these cancer cells are easily to produce resistance to bortezomib. Besides, bortezomib has poor effect in solid tumor treatment. Several19S RP associated deubiquitinases have been shown to be involved in cancer progression and as such are emerging targets for anti-cancer therapies.PSMD7is a deubiquitinase subunit in the lid of19S RP proteasome and occupies a central position within this subcomplex showed by structural organizational analysis. PSMD7is centered by other4subunits forming the heterotetrameric core structure, suggesting the essential role of PSMD7in the lid subcomplex of19S RP. However, the cellular function of it remains unknown. In this study, in order to provide an evidence for the molecular mechanism of proteasome in the ciliary/flagellar disassembly and to find therapy targets for cancer, we investigate the possible function of PSMD7:1. whether is it involved in the ciliary/flagellar disassembly?2. whether does it play a role in tumorigenesis?Part1Proteasome subunit PSMD7is involved in the degradation of the ciliary/flagellar protein kinesinObjectiveThe proteasome activity during flagellar disassembly and interactions of PSMD7with ciliary/flagellar proteins were observed to preliminary study whether PSMD7participated in ciliary/flagellar disassembly and provide basis for further research on the molecular mechanism of UPS in ciliary/flagellar function.Methods1The activity of ubiquitin-proteasome system after flagellar disassemblyImmunofluorescence and Western blotting methods were used to examine the ubiquitin-UNTjunction system in the isolated flagella. After flagellum disassembly induced by IBMX, ubiquitin protein aggregation was observed, the proteasome was purified by glycerol density gradient centrifugation and its activity was determined using fluorescence peptide substrate, and the mRNA expression of PSMD7of D.salina (DsPSMD7) was analyzed by real-time fluorescent quantitative PCR.2The binding of PSMD7with ciliary/flagellar kinesinInteractions of DsPSMD7with KCBP in D.salina cells and HsPSMD7with Kif3a in293T cells were detected by yeast two hybrid technology, which were further UNTfirmed by GST pulldown and co-immunoprecipitation. The degradations of KCBP and Kif3a by proteasome were determined by proteasome inhibitor treatment. Results1The activity of ubiquitin-proteasome system after flagellar disassemblyAfter flagellar disassembly, ubiquitinated proteins were accumulated and the proteasome activity was increased nearly2times, indicating that the ubiquitin-proteasome system is involved in the flagellar disassembly of D.salina. The mRNA level of DsPSMD7was upregulated during flagella disassembly, and reached the highest at30min after disassembly.2The binding of PSMD7with ciliary/flagellar kinesinYeast two hybrid showed that DsPSMD7interacted with KCBP and HsPSMD7interacted with Kif3a, which were UNTfirmed by GST pulldown and co-immunoprecipitation. Two kinesin, KCBP and Kif3a became accumulated in cells after proteasome inhibitor treatment, suggesting that these cilia/flagella kinesin were degraded by proteasome.UNTclusionExpression level of PSMD7, which mediates degradation of the ciliary or flagellar kinesin, is elevated during flagellar disassembly suggesting that PSMD7may participate in ciliary or flagellar disassembly.Part2Functional study of proteasome subunit PSMD7in esophageal squamous cell carcinomasObjective1Differential expression of proteasome between normal esophageal epithelium cells and esophageal squamous cancer cellsThe nucleus, cytoplasm and centrosome were respectively extracted from normal esophageal epithelium and esophageal squamous cancer cells. The translational expressions of PSMD7were detected in various subcellular structures by Western blotting. The localizations of PSMD7were observed by immunofluorescence.2Effect of PSMD7gene knockdown mediated by lentiviral mediated RNAi on the cell proliferation, cycle and apoptosis of esophageal squamous cancer cellsThe accumulation of ubiquitinated proteins and proteasome activity were examined in EC9706cells infected by PSMD7RNAi lentiviral particles. Cell proliferation was analyzed by CCK-8, cell cycle and apoptosis were tested by flow cytometry instrument. The p53level was inspected after PSMD7gene knockdown using Western blotting. The proteasome function and apoptosis of EC9706cells were investigated after PSMD7RNAi lentiviral particles and bortezomib treatment.3The inhibition of xenografted tumor cell growth by PSMD7gene knockdown mediated by lentiviral mediated RNAiXenograft tumor nude mice models were development to explore the tumorigenic capacity of EC9706cells with PSMD7gene knockdown. At the same time, xenograft tumor nude mice were treated by bortezomib for two weeks.The tumor volume was measured and the tumor growth curves were drawn and tumor inhibition rates were calculated. The pathological morphology of tumor cell was observed, the accumulation of ubiquitinated proteins in the tumor cells were surveyed by immunohistochemical,and tumor cell apoptosis in tumor tissues were analyzed by TUNEL method.Results1Differential expression of proteasome between normal esophageal epithelium cells and esophageal squamous cancer cellsThe expression of PSMD7in two of esophageal squamous cancer cells, EC9706and Eca109, were higher than that in normal esophageal epithelium. There was no obvious change in the expressed of it in the nucleus of these three cells, but the esophageal squamous cancer cells had significantly higher expression in the cytoplasm and centrosome than normal esophageal epithelial cells.2Effect of PSMD7gene knockdown mediated by lentiviral mediated RNAi on the cell proliferation, cycle and apoptosis of esophageal squamous cancer cellsThe ubiquitinated proteins were accumulated and the proteasome activity was reduced in EC9706cells after the PSMD7gene knockdown by lentiviral mediated RNAi. PSMD7RNAi also inhibited the proliferation, promoted the apoptosis and affected the function of intracellular p53in EC9706cells. Moreover, the proteasomal functional inhibition and apoptosis induced by bortezomib were enhanced by PSMD7RNAi.3The inhibition of xenografted tumor cell growth by PSMD7gene knockdown mediated by lentiviral mediated RNAiPSMD7RNAi EC9706cells showed lower growth rate in nude mice and the tumor volume of it is only the1/2of the Control group. It had the highest tumor inhibition rate combined with bortezomib therapy with40%tumor volume of the Control group. Pathological histology examination showed reduced tumor cell heterogeneity and increased accumulation of ubiquitinated proteins after PSMD7RNAi and bortezomib treatment. The treatment groups have more apoptosis cells than the Control, in which the combined treatment group showed the best effect in apoptotic induction.UNTclusionPSMD7RNAi can decrease the proliferation, and induce apoptosis of esophageal squamous cancer cells. Also, PSMD7knockdown can effectively inhibit the growth of transplantable tumors in nude mice through inducing apoptosis of tumor cells and raising sensitivity of cancer cells to bortezomib, suggesting that it is likely an effective therapeutic target for esophageal squamous cell carcinoma.