| Objective: Cereblon (CRBN) is widely expressed protein ligase E3,which mediates lenalidomide play anti-proliferative activity in the multiplemyeloma cells and induction T cells to produce cytokines. The study foundlenalidomide need Cereblon to express anti-myeloma activity, and inhibitionof CRBN protein function.CRBN is an important molecular target whenlenalidomide treat with multiple myeloma. This experiment is designed tostudy the immunomodulatory agent lenalidomide and the proteasome inhibitorbortezomib for multiple myeloma cell proliferation, apoptosis and expressionlevels of Cereblon.1Observe the immunomodulator lenalidomide and the proteasomeinhibitor bortezomib inhibits human myeloma cell line RPMI8226proliferation.2Test the immunomodulator lenalidomide and the proteasome inhibitorbortezomib induces apoptosis in myeloma cell line RPMI8226role.3Observe and analyze the effects of the immunomodulator lenalidomideand the proteasome inhibitor bortezomib in human myeloma cell lineRPMI8226of Cereblon (CRBN) gene expression.4Observe and analyze the effects of the immunomodulator lenalidomideand the proteasome inhibitor bortezomib in human myeloma cell lineRPMI8226of Cereblon (CRBN) protein expression.Methods:1First CCK8was used to assay with lenalidomide and bortezomibinhibition growth in RPMI8226.2Flow cytometry was used to detect lenalidomide and bortezomibinduced apoptosis in RPMI8226.3RT-PCR was used to detect lenalidomide and bortezomib impact CRBN gene expression in RPMI8226.4Western Blot was used to detect lenalidomide and bortezomib impactCRBN protein expression in RPMI8226.5The experiment groups were as follows:(1) control group: Do not addany drugs,that lenalidomide concentration is0nmol/L;(2) lenalidomideintervention group:200nmol/L,800nmol/L,3200nmol/L,intervention cells for24h,48h,72h, the best test result was intervention for24h, and48h,72hapoptosis ones, so the following experiments Select intervention for24h todetect lenalidomide amine inhibition of proliferation in RPMI8226cells.(3)bortezomib intervention group:10nmol/L,20nmol/L,40nmol/L concentrationsof bortezomib treated with cells for24h, to detect bortezomib in RPMI8226cell proliferation.6The experimental groups were as follows:(1)control group:do not addany drugs,that lenalidomide concentration is0nmol/L;(2)lenalidomidegroup:200nmol/L,800nmol/L,3200nmol/L concentration of lenalidomidetreated with cells for24h,to detect cell apoptosis in RPMI8226cells.(3)bortezomib group:10nmol/L,20nmol/L,40nmol/Lconcentrations of bortezomibtreated with cells for24h,to detect cell apoptosis in RPMI8226cells.7RT-PCR to detect the influence for CRBN gene expression by differentconcentrations of lenalidomide and bortezomib in RPMI8226cells.8Western Blot to detect the influence for CRBN protein expression bydifferent concentrations of lenalidomide and bortezomib in RPMI8226cells.9Using SPSS13.0statistical software, analysis of variance methodprocessing data. All data with the average of three independent experimentsindicated,that the experimental results are expressed as mean±standarddeviation (X±S); P <0.05considered statistically significant.Results:1Lenalidomide and bortezomib were acting on RPMI8226cells can inhibit their proliferation.(1) respectively200nmol/L,800nmol/L,3200nmol/L concentration of lenalidomide intervention cells for24h; the inhibition rate of each group was (16.67±0.25)%,(26.35±1.21)%,(36.26±1.03) %,dose-dependent,the higher lenalidomide concentration,the higher inhibition rate,the more obvious inhibition,the results were statistically significant (P=0.00)(Fig.1,Table1).(2)respectively10nmol/L,20nmol/L,40nmol/L concentration of bortezomib intervention cells for24h; the inhibition rateof each group was (8.13±1.14)%,(18.46±0.21)%,(31.43±1.64)%; dose-dependent, the higher bortezomib concentration, the higher inhibition rate,themore obvious inhibition, the results were statistically significant (P=0.00)(Fig.2, Table2).2Lenalidomide and bortezomib effect pro-apoptotic in RPMI8226cells. Flow cytometry results showed that:(1) respectively0nmol/L,200nmol/L,800nmol/L,3200nmol/L concentration of lenalidomide intervention cells for24h, the apoptotic rate was (7.12±0.59)%,(17.89±0.31)%,(29.4±0.71)%,(40.33±1.25)%; in a dose-dependent manner, the higher lenalidomide concentration, the higher apoptosis rate, the more obvious pro-apoptotic, the results were statistically significant (P=0.00)(Fig.3, Fig.5,Table3).(2) respectively0nmol/L,10nmol/L,20nmol/L,40nmol/L bortezomib intervention cells for24h, the apoptotic rate was (7.82±0.33)%,(14.26±0.60)%,(25.22±0.26)%,(34.54±1.24)%,dose-dependent,the higher bortezomib concentration, the higher apoptosis rate, the more obvious pro-apoptotic,the results were statistically significant (P=0.00)(Fig.4, Fig.6, Table4).3Lenalidomide and bortezomib impact on CRBN gene expression.Real-time PCR results showed that:(1) respectively0nmol/L,200nmol/L,800nmol/L,3200nmol/L concentration of lenalidomide intervention cells for24h, CRBN gene expression levels were1,0.81±0.02,0.52±0.01,0.23±0.02. The CRBN gene expression level in a dose-dependent manner, with the increase of lenalidomide concentration, reduced expression,the results were statistically significant (P=0.00)(Fig.7, Fig.8, Table5).(2) respectively0nmol/L,10nmol/L,20nmol/L,40nmol/L concentration of bortezomib intervention cells for24h,CRBN gene expression levels were1,0.39±0.03,0.17±0.02,0.09±0.02. The CRBN gene expression level in a dose-dependent manner, with the increase of bortezomib concentration, reduced expression,the results were statistically significant (P=0.00)(Fig.9, Fig.10,Table6).4Lenalidomide and bortezomib impact on CRBN protein expression. Western Blot results showed that:(1) respectively0nmol/L,200nmol/L,800nmol/L,3200nmol/L concentration of lenalidomide intervention cellsfor24h, CRBN protein expression levels were1.48±0.03,1.14±0.01,0.95±0.01,0.56±0.02. The CRBN protein expression in a dose-dependent manner, with the increase of lenalidomide concentration, reduced expression, the results were statistically significant (P=0.00)(Fig.11, Table7).(2) respectively0nmol/L,10nmol/L,20nmol/L,40nmol/L concentration of bortezomib intervention cells for24h, CRBN protein expression levels were1.39±0.1,1.32±0.1,1.36±0.2,1.34±0.2. The results showed that bortezomib had not effect on the expression of CRBN protein,and were not statistical significance (P=0.92)(Fig.12, Table8).Conclusion:1Lenalidomide inhibition in myeloma RPMI8226cells showed a dose-dependent manner，with increasing lenalidomide concentration, the inhibitionrate increased. bortezomib inhibition in myeloma RPMI8226cells showed adose-dependent manner, with increasing bortezomib concentration, theinhibition rate increased.2Lenalidomide induced apoptosis in myeloma RPMI8226cells showeda dose-dependent manner, with increasing lenalidomide concentration, theapoptosis rate increased. Bortezomib induced apoptosis in myelomaRPMI8226cells showed a dose-dependent manner, with increasingbortezomib concentration, the apoptosis rate increased.3Lenalidomide inhibited the CRBN gene expressing in myelomaRPMI8226cells showed a dose-dependent manner, with increasinglenalidomide concentration, expression reduced. Bortezomib inhibited theCRBN gene expressing in myeloma RPMI8226cells showed a dose-dependent manner, with increasing bortezomib concentration, expression reduced.4Lenalidomide inhibited the CRBN protein expressing in myelomaRPMI8226cells showed a dose-dependent manner, with increasinglenalidomide concentration, expression reduced. Bortezomib had not effect onthe expression of CRBN protein in RPMI8226myeloma cell. |
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