| Mltipel Myeloma (MM) is a still incurable disease of the blood with high mortality and relapse rates. Extensive molecular and genomic characterization of signal molecules and pathways of MM cells will provide novel drugable targets for its targeting therapy. SUMO/sentrin specific protease 1 (SENP1) is an important regulation protease in the protein sumoylation, which affects the cell cycle, proliferation and differentiation. The role of SENP1 and is mediated protein desumoylation in pathopHsiology of multiple myeloma is unknown.Objectives:(1) Investigating the expression status of SENP1 in multiple myeloma cells (2) Measuring the effect of SENP1 silence on apoptosis,proliferation,cell cycle and the ability of colony formation of multiple myeloma cells.(3)Further research of the mechanism of SENP1 konckDown interacts with the important, aberrant pathway in multiple myeloma.Methods:(l) Using healthy donors’ bone marrow mononuclear cells as control, employing real-time PCR and western blot assays to detect the expression of SENP1 in 3 kinds of multiple myeloma cell lines and 6 cases of MM patients’ CD138+ cells.(2) Using lentivial vector contain short hairpin RNA (shRNA) to infect MM cell lines for 48h, employing flow cytometer and fluorescence microscope to measure the efficiency of infection,and at the same time, using QPCR, western blot to confirm the efficiency of knocking out.(3) Using MM cells which infected by control virus, employing lentivirus shRNA to knock out SENP1 and then using CCK8 to detect the proliferation status of MM cell lines in different time points. (4)After infecting lentivirus shRNA for 48h,using cells which was infected control virus as control,employing flow cytometer to detect the change of apoptosis and cell cycle in MM cells.(5) Using western blot to confirm the efficiency of IL-6’s induction of SENPl,the interaction between SENP1 and NF-κ B and the impact of SENP1 koncking out on the IL-6 activating NF-κ B,and using EMS A to further detect the effect of SENP1 knocking out on NF-κ B’s transduction activity.Results:we find that the percentage of apoptosis increased about 40% in MM cell lines XG-7 and RPMI8226.CCK8 assay were applied to detect the change of proliferation after SENP1 was knocked out in XG-7 and RPMI8226.When SENP1 was knocked out for 48h, using flow cytometer to detect cell cycle and find that XG-7’s apoptosis was induced and RPMI-8226’s percentage of G0/G1 was significantly increased.After SENP1 was knocked out, treating with Bortezomib and observed that the killing effect was increased.(3) Further investigation of mechanism we find that IL-6 can induces the expression of SENP1, in time- and dose-dependent fashion,during this process, STAT3 was in upstream of SENP1.Koncking out SENP1 can inhibits the pHospHorylation of P65 and IKB a in NF- κB pathway,and block NF- κ B’s inhibitor IKBa’s degradation,the level of SUMO-2 binding total protein was increased at the same time, which can explain the inhibition of NF-κB after SENP1 was knocked out. Interestingly, bortezmib treatment inhibits NF-κB pathway and find that decrease of the expression of SENPl,indicated that cycle fashion of SENP1 and NF-κB interaction.Conclusions:SENP1 was highly expressed in MM cell lines and primary cells,knocking out SENP1 caused not only the apoptosis of MM cell lines,but also the inhibition of proliferation and the change of cell cycle,effect of killing of bortezomib was also increased.This process was related to the interaction of among SENP1,IL-6 and NF-κ B.Our research provide a new target of thrapy of MM,make a foundation of further in vivo experiment and clinical research in the future. |
