来那度胺诱导多发性骨髓瘤细胞凋亡机制

Mechanism of multiple myeloma cell apoptosis induced by lenadomide

目的探讨来那度胺通过降解转录因子伊卡洛斯家族锌指蛋白(Ikaros family zinc finger,IKZF)1、IKZF3诱导多发性骨髓瘤细胞凋亡的机制。方法对数生长期人源多发性骨髓瘤细胞株RPMI 8226细胞分为0μmol/L组、1μmol/L组、2.5μmol/L组、5μmol/L组、10μmol/L组、20μmol/L组,分别添加浓度为0、1、2.5、5、10、20μmol/L来那度胺至细胞培养基中,培养24h后采用台盼蓝染色法测定各组细胞活力,采用流式细胞术检测各组细胞凋亡情况,应用实时PCR检测各组细胞CRBN mRNA表达水平,采用ELISA法检测各组细胞内IKZF1、IKZF3、干扰素调节因子4(interferon regulatory factor 4,IRF4)、白细胞介素-2(interleukin-2,IL-2)蛋白表达水平。结果 0μmol/L组、1μmol/L组、2.5μmol/L组、5μmol/L组、10μmol/L组、20μmol/L组的RPMI 8226细胞生存率[(100.00±0.00)%、(98.25±0.63)%、(80.02±2.37)%、(57.24±4.55)%、(22.86±4.13)%、(18.41±3.06)%]依次降低,细胞凋亡率[0、(1.88±0.21)%、(18.64±3.50)%、(41.04±5.62)%、(70.49±10.11)%、(79.70±12.35)%]依次升高,CRBN mRNA上调倍数(0、0.68±0.11、1.35±0.24、2.23±0.30、2.99±0.35、3.21±0.38)依次升高,IKZF1[(36.75±3.04)、(33.18±3.11)、(30.55±3.84)、(28.71±2.18)、(25.60±2.05)、(20.44±1.84)μg/L]、IKZF3[(41.04±3.13)、(38.26±3.45)、(33.60±3.02)、(30.58±2.87)、(22.03±2.22)、(19.66±2.15)μg/L]、IRF4[(12.30±1.65)、(11.66±1.33)、(10.24±0.87)、(8.35±1.21)、(6.04±1.03)、(5.74±1.00)μg/L]和IL-2[(8.63±1.21)、(8.02±1.15)、(6.80±0.96)、(5.34±0.69)、(4.87±0.52)、(4.22±0.37)μg/L]水平依次降低,组间比较差异均有统计学意义(P<0.05)。结论来那度胺可调节CRL4-CRBN复合物的活性,增加转录因子IKZF1和IKZF3的泛素化,降低IKZF1和IKZF3蛋白活性,下调IRF4、IL-2蛋白表达水平,最终诱导多发性骨髓瘤细胞凋亡。

Objective To investigate the mechanism of multiple myeloma cell apoptosis by lenadomide via degrading transcription factors Ikaros family zinc finger(IKZF)1 and IKZF3.Methods RPMI8226 cells in logarithmic growth phase were divided 0,1,2.5,5,10 and 20μmol/L lenalidomide groups according to the concentrations after 24 h culture.Trypan blue staining was used to detect the activity of RPMI 8226 cell,flow cytometry was used to detect the apoptosis of RPMI8226 cells,real-time PCR was used to detect the expression level of CRBN mRNA,and ELISA was used to detect the expressions of IKZF1,IKZF3,interferon regulatory factor 4(IRF4)and interleukin-2(IL-2)proteins in all groups.Results The survival rates of RPMI 8226 cells reduced gradually((100.00±0.00%,(98.25±0.63)%,(80.02±2.37)%,(57.24±4.55)%,(22.86±4.13)%,(18.41±3.06)%)(P<0.05),the apoptosis rates increased gradually(0,(1.88±0.21)%,(18.64±3.50)%,(41.04±5.62)%,(70.49±10.11)%,(79.70±12.35)%)(P<0.05),the expressions of CRBN mRNA increased gradually(0,0.68±0.11,1.35±0.24,2.23±0.30,2.99±0.35,3.21±0.38)(P<0.05),while IKZF1((36.75±3.04),(33.18±3.11),(30.55±3.84),(28.71±2.18),(25.60±2.05),(20.44±1.84)μg/L),IKZF3((41.04±3.13),(38.26±3.45),(33.60±3.02),(30.58±2.87),(22.03±2.22),(19.66±2.15)μg/L),IRF4((12.30±1.65),(11.66±1.33),(10.24±0.87),(8.35±1.21),(6.04±1.03),(5.74±1.00)μg/L)and IL-2((8.63±1.21),(8.02±1.15),(6.80±0.96),(5.34±0.69),(4.87±0.52),(4.22±0.37)μg/L)decreased gradually(P<0.05)in 0,1,2.5,5,10 and 20μmol/L lenalidomide groups in turns.Conclusion Lenalidomide induces apoptosis of multiple myeloma cells by regulating the activity of CRL4-CRBN complex,increasing the ubiquitination of transcription factor IKZF1 and IKZF3,decreasing the activities of IKZF1 and IKZF3 protein,and downregulating the expressions of IRF4 and IL-2.