The Effect Of SENP1 On The Biological Characteristics Of NK92 Cells

Natural killer(NK)cells are a type of lymphocyte and an important component of innate immune system.Their biological characteristics including proliferation,maturation and survival are regulated by both the intracellular and extracellular signal molecules.The cell signaling molecules are regulated by protein phosphorylation and post-translational modification.SUMOylation(Small ubiquitin-like modifier protein(SUMO)modification),a type of key regulatory modifications with small ubiquitin-related modifier,may affect the functional activity of signal networks.SUMO specific protease 1(SENP1)is an important protease that regulates the SUMOylation of proteins and affects cell cycle,proliferation and differentiation.SENP1 deconjugates SUMO from sumoylated proteins and regulates SUMO pathways.It is known that SENP1 affect erythropoiesis by regulating of HIF-1? sumoylation,but its effects on the biological characteristics of NK cells is not clear.IL-21 is a newly found cytokine,mainly produced by the activation of CD4+ cells.IL-21 receptor are expressed in the resting and activated NK cells and play an important role in regulating cell proliferation and cell function.But whether SENP1 is involved in IL-21 signal transduction and regulation of NK cell biological characteristics has not been studied.Objectives:(1)To clarify the biological function role of IL-21 in regulation of SENP1 expression in NK92 cells.(2)To investigate the effect of interfering Senp1 on apoptosis,proliferation of NK92 cells.(3)To determine the effect of interfering Senp1 on cytotoxicity ability of NK92 cells.Methods:(1)The NK 92 cells were deprived of IL-2 in culture and stimulated with IL-21.The cell proliferation was determined by using CCK8 assay.The apoptosis of NK92 cells were determined by applying Annexin V-PI double labeling.The expression of SENP1 in NK92 cells were detected by employing real-time quantitative polymerase chain reaction(q RT-PCR)and western blot.(2)Using lentiviral vector carrying Senp1 short hairpin RNA(sh RNA)to infect NK92 cells for 48 h,employing flow cytometer and fluorescence microscope to measure the efficiency of infection and using q RT-PCR,western blot to confirm the efficiency of knockout.(3)After infecting lentivirus sh RNA for 48 h,using cells which was infected control virus as control,employing lentivirus sh RNA to knock out Senp1 and then using CCK8 to detect the proliferation status of NK92 cells in different time points and employing flow cytometer to detect the change of apoptosis in NK92 cells.(4)After infecting lentivirus sh RNA for 24 h and 48 h,using q RT-PCR to detect the expression of cytotoxic factors in NK92 cells.(5)The cytotoxicity of NK92 cells against K562 cells was detected by luciferase reporter gene assay.Results:(1)Treatment of NK92 cells with IL-21 results in upregulated SENP1 expression in a concentration-dependent fashion.It is indicated that IL-21 is an inducer of SENP1 expression.(2)The NK92 cells undergo apoptosis after deprivation of IL-2.IL-21 treatment reduces the apoptosis of NK92 cells induced deprivation of IL-2.(3)Transfection with Lentivirus-mediated sh RNA significantly decreased expression of SENP1 in NK92 cells both at m RNA and protein level.(4)The apoptosis of Senp1 sh RNA transduced cells were determined by Annexin V assay.the percentage of apoptotic cells of Senp1 interference group was significantly increased compared with control group at 24 h post transduction.It is indicated that Senp1 interference promotes apoptosis of NK92 cells.(5)CCK8 assay were applied to detect the proliferation of NK 92 cells.Senp1 knockdown significantly inhibits the proliferation of NK92 cells.(6)Interestingly,The cytotoxicity ability of NK92 cells against K562 cells was increased in senp1 sh RNA group compared to that of control group.The expression levels of cytotoxic factors Granzyme B and perforin were upregulated,whereas IFN-? was down regulated.Conclusions: SENP1 is an important protease that regulates the SUMOylation of proteins and affects cell survival and proliferation of NK cells.IL-21 treatment obviously increases the expression of SENP1 in NK92 cells.It inhibits the apoptosis and reduces the number of NK92 death caused by IL-2 deprivation.SENP1 knockdown induces apoptosis and inhibits cell proliferation,and affects the cytotoxicity of NK92 cells in killing K562 cells.