The Effects And Mechanisms Of SUMO Ylation On The Wnt/?-catenin Pathway In Myeloma Cells

[Background and Objective]Multiple myeloma is a malignant plasma cell disease, and is currently the second most common hematologic malignancies. It is characterized by monoclonal immunoglobulinin the blood and urine, with pathogenesis remains unclear. The disease has no cure, almost all multiple myeloma patients will ultimately relapse.Although patients may benefit from novel protease inhibitors and immunomodulatory agents, the drug resistance is becoming an increasingly serious problem for myeloma treatment. A deep understanding of the pathogenesis of multiple myeloma has become an imperative need for myeloma treatment. SUMO modification is an important post-translational modification of proteins, with the ability of modifying the biological functions of substrate proteins. Compared with normal plasma cells, the degree of SUMO modification significantly increased in myeloma patients, and associated with poor prognosis, with the exact mechanism clear.Our previous study found that the classic Wnt / ?-catenin pathway is highly active in multiple myeloma, and can promote the proliferation and drug resistance of myeloma cells. To explore whether the SUMO modification of Wnt / ?-catenin pathway would influence the biological characteristics of myeloma, we used myeloma cell lines NCI-H929 and RPMI-8226 as the research object. SUMO-1small interfering RNA was used as a tool to detect the effect of SUMOylation inhibition on Wnt pathway and its specific mechanism. Experiment is divided into four parts. For the first part, we detect the inhibition of SUMOylation modification on Wnt/?-catenin pathway activity and the downstream genes. For the second part,we detect the inhibition of SUMOylation modification on molecule ?-catenin and the molecular mechanisms. For the third part, the interaction of SUMO-1 and ?-catenin in myeloma cells was examined.[Methods]1. The effect of SUMOylation inhibition on the activity of the Wnt/?-catenin pathway and its downstream molecules.Order SUMO-1 si RNA synthesis based on the literature and the results of preliminary experiments. Liposomal transfection method(lipo2000) was used to transfect SUMO-1 si RNA into the myeloma cell line(RPMI-8226, NCI-H929).Real-time PCR and Western blot were used to test the efficiency of interference. The one with highest interference efficiency was selected. Luciferase reporter gene was used to detect Wnt/?-catenin pathway activity. Real-time PCR and Western blot were used to detect the expression of c-Myc, cyclin D1 and survivin.2. The inhibition of SUMOylation modification on molecule ?-catenin and the molecular mechanisms.Using Real-time PCR and Western blot to detect ?-catenin expression levels before and after the SUMO-1 interference. Cells were treated with 100?g / ml CHX(cycloheximide), collected and lysed at different time points(0,0.5,1,1.5 hours). The myeloma cells were collected and precipitated using anti-?-catenin antibody and anti-Ubiquintin antibody respectively. By liposomal transfection method, myeloma cells were transfected with SUMO-1 si RNA and ?-catenin(WT) or ?-catenin(S33Y).After 48 hours, the luciferase reporter activity was detected. Flow cytometry(PI /Annexin V staining) and MTS method were used to detect apoptosis and proliferation of myeloma cells.3. The interaction of SUMO-1 and ?-catenin in myeloma cellsThe multiple myeloma cells NCI-H929 and RPMI-8226 were lysed and immunoprecipitat using anti-?-catenin antibody. Then the proteins were detected with anti-?-catenin and anti-SUMO-1 antibodies respectively. The NCI-H929 cells were fixed, blocked, and incubated with anti-?-catenin and anti-SUMO-1 antibodies overnight. Then DNA was stained using DAPI. Corresponding fluorescent secondary antibodies were incubated for 2 hours. The cells were finally detected by fluorescence confocal microscope.[Results]1. The effect of SUMOylation inhibition on the activity of the Wnt/?-catenin pathway and its downstream molecules.At the m RNA level, the third fragment of SUMO-1 si RNA was with inhibition efficiency of> 80%. At the protein level, the third fragment of SUMO-1 si RNA was with inhibition efficiency of> 70%. In both myeloma cell lines, the Wnt/?-catenin pathway activity was significantly decreased after SUMO-1 inhibition. SUMO-1si RNA treatment decreased the expression of Wnt/?-catenin pathway downstreammolecules c-myc, cyclin D1 and survivin.2. The inhibition of SUMOylation modification on molecule ?-catenin and the molecular mechanisms.The results showed that m RNA levels of ?-catenin in the SUMO-1 interference group and the control group remained unchange. The ?-catenin protein levels in SUMO-1 si RNA-treated group were significantly reduced. With the increase of SUMO-1 si RNA interference strength, the ?-catenin expression levels also decreased in a dose-dependent manner. SUMOylation inhibiton increased the ?-catenin ubiquitination degree. Overexpression of ?-catenin(S33Y) plasmid can partially reverse Wnt/?-catenin pathway activity after SUMOylation inhibition.3. The interaction of SUMO-1 and ?-catenin in myeloma cellsIn two myeloma cell lines, SUMO-1 binds to the endogenous ?-catenin. In fluorescence confocal microscope, we found ?-catenin co-localizd with SUMO-1.Suppression of SUMOylation modification can significantly reduce the SUMO modification of ?-catenin. In primary myeloma cells, SUMO-1 binds to the endogenous ?-catenin.?Conclusions?1. SUMO modification of ?-catenin activated the Wnt/?-catenin pathway.2. SUMO modification affects the biological activity of myeloma cells via the Wnt/?-catenin pathway.