| Objective:The present study is designed to investigate the effect of BET Inhibitor OTX015 against multiple myeloma via targetting the important oncogene c-MYC and it,s potential mechanism.Methods:(1)The multiple myeloma cell lines MM1S and RPMI8226 were selected as the experimental objects.Cell proliferation was determined by CCK8 assay,The IC50(half maximal inhibitory concentration)values was calculated according to the results of CCK8 assay.For cell cycle and apotosis analysis,each cell line was treated with a range of OTX015 concentration designed according to IC50 values for 72h..Each cell line was treated with the concentration of OTX015 designed based on the IC50 values for 4h、8h、24h,then real-time reverse transcription quantitative polymerase chain(RT-qPCR)and western blot analysis were performed to detect the expression change of c-MYC at the level of mRNA and protein.(2)Mononuclear cells were isolated from bone marrow of selected multiple myeloma patientsand cultured in Low-glucose DMEM medium supplemented with 10%fetal bovine serum to get MSCs,The culture-expanded cells were cultured in an osteogenic induction medium,OTX015 was mixed into the medium at final concentration of 500nM/L for the experimental plate,Plate with untreated cells wasused as vehicle controls.At the timepoint of 14 days.we performed Von Kassa stain to reveal osteogenic differentiation of calcinosis.RT-qPCRwas performed to test the change of osteogenic-related genes OSX,OCN and Runx2(3)For vivo experiments,NPG mouses were injected with RPMI8226 cells through caudal vein,Small Animal Imaging Technology was performed 3 weeks after injection to assess the tumor burden,selected mouses were separated into two cohort,one cohort as experimental subjects while another as the vehicle,each cohort contained 8 mouses.Experimental subjects were treated orally with OTX015 75mg/kg daily.After the treatment,seleceted animals from two cohort were employed to get serum and femur.Serum was used to assess PINP(bone formation)and CTX(bone resorption)by ELISA.MicroCT was used to check Femoral epiphysis’varation.(4)CD138~+cells were isolated from bone marrow of 9 primary multiple myeloma patients,then treated with OTX015 for 12h,RT-qPCR was performed to assess the expression of c-MYC.Results:(1)OTX015 suppressed the growth of myeloma cell lines significantly.IC50of MM1Sand RPMI8226 was 39.16nmol/L and 1600nmol/L respectively.Flow cytometry for cell cycle analysis detected significant arressment in G0/G1 phase,and deep supresstion of S and G2/M phase.While no marked apotosis was detected.The m RNA and protein level of c-MYC decreased obviously depending on the time point.(2)Von kassa staining showed enhanced mineral deposition of MSCs treated with OTX015 compared with vehicle sujects after osteogenic differentiation for two weeks.RT-qPCR revealed treated MSCs expressed enhanced m RNA levels of osteogenic markers OSX,OCN,Runx2 compared with vehicle subjects.(3)In vivo experiments,OTX015-treated cohort expressed enhanced PINP(bone formation)factor but weaked CTX-I(bone resorption)compared with the vehicle cohort.MicroCT analysis revealed much more severe bone structure damage of untreated subjects compared with OTX015-treated subjects.(4)OTX015 significantly inhibited CD138 active cell activity,OTX015 treatment to primary multiple myeloma patients’CD138 active cells leads to weakend m RNA expression level of c-MYC than the untreated subjects.Conclusions:OTX015 can significantly inhibit the growth of myeloma cells;OTX015can significantly reduce the expression of c-MYC in myeloma cell line.OTX015 can promote bone formation by promoting calcium deposition,thereby combating myeloma bone destruction.OTX015 inhibits the activity of CD138+in patients with newly diagnosed multiple myeloma and significantly reduces the expression of c-MYC. |
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