| Objective: To investigate the effects of the third-generation immunomodulatory pomalidomide on proliferation,apoptosis,cell cycle,immunomodulator binding protein CRBN and ERs-related mRNA and protein expression in human multiple myeloma cell line MM1.S.To investigate the regulatory mechanism of pomalidomide on CRBN protein and ERs-related molecules in MM1.S cells.Methods: The multiple myeloma MM1.S cells were used as the research object.After resuscitation,semi-suspended semi-adherent cells were obtained and the cell morphology was observed.The cell growth curve was drawn by manual cell counting in logarithmic growth phase.Cell proliferation was detected by CNK-8 method after pomalidomide treatment,and apoptosis and cycle were detected by flow cytometry.The follow-up experiment is divided into two parts: Experiment 1(Impact of pomalidomide on CRBN mRNA and protein expression): According to the concentration of pomalidomide,the expression of CRBN in MM1.S cells at 24 h,48h and 72 h was detected by RT-PCR and Western blot.Experiment 2(The effect of pomalidomide on the mRNA and protein expression of ERs-related molecules(GRP78/BiP,XBP-1s and CHOP)): According to the action time of 40umol/L and/or 80umol/L pomalidomide,it was divided into 8 groups(0h,4h,8h,12 h,16h,24 h,48h and 72h),the mRNA and protein expression of ERs-related molecules was detect by RT-PCR and Western blot dividedly.Results:1.The MM1.S cells after resuscitation and passage were round and semi-suspended and semi-adherent;The size of the cell body is about 15-20?M,the nucleus is round,the nuclear chromatin is arranged in coarse granular shape,the nucleolus is faintly visible,2-4,the cytoplasm is moderate,and it is opaque blue-gray,foamy,visible pseudopod;In thelogarithmic growth phase,the cells form a cluster and resemble a "grape bunch",which is easily blown into a single cell suspension state by a pipette.2.CCK8 assay showed that compared with the 20umol/L group,the inhibition rate of cell proliferation at 24 hours,48 hours and 72 hours increased with the increase of POM concentration(P<0.05);Compared with 24 hours,the cell inhibition rates of 20umol/L group,40umol/L group and 80umol/L group increased with the prolongation of POM time(P<0.05).3.Flow cytometry was used to detect MM1.S cell apoptosis: Compared with the control group,the proportion of early apoptotic cells in each group increased with the increase of POM concentration at 24 hours,48 hours and 72 hours(P<0.05);Compared with 24 hours,the proportion of early apoptotic cells increased with the prolongation of POM action time(P<0.05).4.Flow cytometry to detect MM1.S cell cycle: Compared with the control group,the proportion of cells in G0/G1 phase increased with the increase of POM concentration in 24 hours,and cell proliferation was inhibited(P<0.01);The proportion of cells in G0/G1 phase increased only in 80umol/L group in 48 hours,and cell proliferation was inhibited(P<0.01);There was no significant difference in the proportion of cells in each group at 72 hours,and cell proliferation was not significantly affected(P>0.05).5.RT-PCR was used to detect the expression of CRBN mRNA: Compared with the control group,the relative expression of CRBN mRNA decreased with the increase of POM concentration in 72 hours,and the difference was significant in 40umol/L and 80umol/L groups(P<0.05).6.Western blot was used to detect the expression of CRBN protein: Compared with the control group,the expression of CRBN protein decreased with the increase of POM concentration in 72 hours,and the difference was significant in the 80umol/L group(P<0.05).7.RT-PCR was used to detect the expression of ERs-related molecular mRNA:(1)XBP-1 mRNA expression: 80?mol/L POM was applied to MM1.S cells,and the expression of XBP-1 mRNA decreased gradually from 0 to 12 hours(P<0.05),gradually increasing from 16 to 24 hours and rose to the highest(P<0.05)and gradually decreased at48-72 hours(P<0.05).(2)GRP78/BIP mRNA expression: 80?mol/L POM was applied to MM1.S cells,and GRP78/BIP mRNA expression decreased gradually from 0-12 hours(P<0.01),gradually increasing from 16 to 24 hours and rose to the highest level(P<0.01)and gradually decreased in 48-72 hours(P<0.01).(3)CHOP mRNA expression: 80?mol/L POM effect MM1.S cells,with the prolongation of time,the expression of CHOP mRNA was relatively stable at 0-16 hours,gradually increased after 24 hours,reached the highest level at 72 hours(P <0.01).8.DddsWestern blot analysis of ERs-related protein expression:(1)Expression of XBP-1s protein: 40umol/L POM was applied to MM1.S cells,and the expression of unspliced XBP-1u and spliced XBP-1s increased with the increase of POM time(P<0.01),and both of them reached the highest level in 24 hours;XBP-1u increased significantly,and the expression of XBP-1u and XBP-1s decreased after 24 hours,but it was still higher than 0 hours(P<0.05).(2)Expression of GRP78/BIP protein: 40umol/L POM was applied to MM1.S cells,the expression of GRP78/BIP protein increased gradually from 0-24 hours with the prolongation of action time,and reached the highest level at 24 hours(P<0.01),and gradually decreased after 24 hours,but still higher than the 0 hour group(P < 0.05).(3)Expression of CHOP protein: 0umol/L,40umol/L and 80umol/L POM were applied to MM1.S cells for 72 hours,with the increase of POM concentration,the expression of CHOP protein increased gradually(P<0.05);40umol/L POM acted on MM1.S cells,the expression of CHOP protein gradually increased with the prolongation of POM action time,reaching the highest level at 72 hours(P<0.01).Conclusions:1.Pomhexamine inhibited the proliferation of MM1.S cells,induced apoptosis,arrested cell cycle in G0/G1 phase,and decreased the expression of CRBN mRNA and protein in a concentration-and time-dependent manner.Therefore,CRBN is essential for Pomalidomide to fight multiple myeloma.2.Pomalidomide caused the mRNA and protein expression of the endoplasmic reticulum stress marker protein GRP78/BIP and the unfolded protein response pathway-relatedprotective protein XBP1 in MM1.S cells to increase first and then decrease;The mRNA and protein expression of the unfolded protein response pathway-related injury protein CHOP increased in a concentration-and time-dependent manner;Therefore,pomalidomide can induce endoplasmic reticulum stress in MM1.S cells and participate in the regulation of cell damage through its downstream unfolded protein response. |
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