Effects Of DJ-1 Inhibition On The Proliferation,Apoptosis,and Cell Cycle Progression Of Multiple Myeloma Cells

Objectives:1.The expression of DJ-1 mRNA and protein in human multiple myeloma cell lines(HMCLs);2.Effects of DJ-1 on the proliferation,cell cycle progression and apoptosis of two representative HMCLs(RPMI8226 and U266);3.To investigate whether DJ-1 is associated with RPMI8226,U266 cells’ response to drugs-(bortezomib,dexamethasone)induced apoptosis.Methods:1.Real time fluorescent quantitative polymerase chain reaction(RT-qPCR)and Western Blot(WB)were used to detect the DJ-1 mRNA and protein expression in RPMI8226,U266;2.RPMI8226 and U266 cells were infected with the control shRNA lentiviral particles-A and DJ-1 shRNA(h)lentiviral particles,then the stable transfected cells were screened by being treated with medium containing puromycin of an appropriate concentration,and the inhibition of DJ-1 was validated by mRNA and protein detection;3.RPMI8226 and U266 cells were transfected with lentiviral vector to knockdown DJ-1 expression.Cell proliferation,apoptosis and cell cycle changes were compared between groups by using CCK8 colorimetric assay and flow cytometry(FCM);4.Lentiviral vector were used to knockdown the expression of DJ-1 in RPMI8226 and U266 cells,and then cells were treated with bortezomib and dexamethasone.Next,cell proliferation was compared with control cell linesby using CCK8 colorimetric assay,and cell cycle changes and apoptosis were compared with control cell lines by using FCM analysis.Results:1.Compared with the human peripheral blood mononuclear cells(PBMCs),both RPMI8226 and U266 cells had higher levels of DJ-1 mRNA and protein expression.2.RPMI8226 and U266 were transfected with DJ-1 shRNA lentiviral particles(shDJ-1),and cells which had a stable DJ-1 inhibition were screened by being treated with puromycin-containing culture medium.As a result,the expression of DJ-1 mRNA and protein were significantly lower than those in the blank control and shControl groups,which indicated that a myeloma cell model with DJ-1 inhibition was successfully constructed.3.Compared with shControl cells,cells transfected with shDJ-1 had a significantly decreased proliferation rate(P<0.05),but except for the two groups of RPMI8226 cells after 48 hours’ culture(OD values were no significant difference)(P=0.0779);similarly,the percentage of cells in G1 phase were decreased and S phase were increased in cells transfected with shDJ-1,suggesting that these cells were arrested in S phase(P<0.05);at last but not at least,the early apoptosis rate of cells were significantly higher in shDJ-1 groups(P<0.05).4.Each group of cells were treated with indicated concentrations of bortezomib(final concentration 0,2.0,4.0,6.0,8.0,10.0nmol/L)or dexamethasone(final concentration 0,0.5,1.0,5.0,10.0,20.0μmol/L)for 24 hours,and then the inhibition rate of cell proliferation was calculated according to the OD values measured by using CCK8 colorimetric assay.As a result,we found that the inhibition rate of cell proliferation was increasedwith increasing drug concentrations;under a fixed drug concentration,proliferation inhibition rate of shDJ-1 cells was significantly higher than that of shControl cells(P<0.05),with an exception of U266 cells treated with dexamethasone of 0.5 or 1.0μmol/L,in which no statistically significant difference was found between shDJ-1 and shControl cells.5.Each group of RPMI8226 cells were treated with bortezomib(final concentration of 2.0nmol/L)or dexamethasone(1.0μmol/L)for 48 hours,and then flow cytometry was used to analyze the cell cycle progression,and we found that the proportions of shDJ-1 cells in G1 and S phase were both significantly higher in shDJ-1 cells than those of shControl cells(P<0.05),suggesting that MM cells with DJ-1 inhibition were blocked in S phase under the treatment of indicated agents.6.Cells were treated with grading concentrations of bortezomib(final concentrations of 0,2.5,5.0,10.0nmol/L)and dexamethasone(final concentrations of 0,0.1,1.0,10.0μmol/L)for 24 hours and cells were harvested for Annexin V/PI staining,followed by an apoptosis assay with flow cytometry.We found that early apoptosis rate of MM cells increased with increasingly grading concentrations of agents;under a fixed concentration of agents,the apoptosis rate of shDJ-1 cells were significantly higher than that of shControl cells(P<0.05),except for that of U266 cells treated with 5.0nmol/L of bortezomib or 0.1μmol/L of dexamethasone.Conclusions:1.DJ-1 mRNA and protein expression are abnormally elevated in two representative human multiple myeloma cell lines(RPMI8226 and U266).2.DJ-1 is involved in the regulation of cell proliferation,cell cycle progression and apoptosis in RPMI8226 and U266 cells,and DJ-1 inhibitionis able to enhance the sensitivity of MM cells to bortezomib and dexamethasone,but the associated mechanisms need further investigations.3.DJ-1 may be a candidate target for MM treatment,deserving a further study.