| Part 1.mi R-221/222 regulate the sensitivity of multiple myeloma cells to dexamethasoneObjective: To investigate the expression levels of mi R-221/222 in multiple myeloma(MM)cells and evaluate their role in drug resistance.Methods: q RT-PCR analysis was used to detect the expression levels of mi R-221/222 in CD138+ cells from MM patients and healthy individuals and then the difference of mi R-221/222 expression between MM patients and healthy individuals was analyzed.The expression levels of mi R-221/222 in human myeloma cell lines(HMCLs)were detected by q RT-PCR and the sensitivity of these cell lines to dexamethasone(Dex)was evaluated by CCK-8 assay.Dex-sensitive MM cells were transfected with agomir-221/222 or agomir-NC and Dex-resistant MM cells were transfected with antagomir-221/222 or antagomir-NC and then were treated with Dex at the indicated concentrations,cell viability was measured using the CCK-8 assay.Results: Compared with healthy controls,the expression levels of mi R-221 and mi R-222 were markedly upregulated in plasma cells(PCs)from patients with newly diagnosed MM,and they were further upregulated in PCs from patients with relapsed or refractory MM.In addition,MM.1S,U266,JJN3,and RPMI-8226 were identified as Dex sensitive,while MM.1R,ARH-77,NCI-H929,and MR-20 were Dex resistant.The expression levels of mi R-221 and mi R-222 were relatively higher in the Dex-resistant HMCLs than in the Dex-sensitive HMCLs.Moreover,overexpressions of mi R-221/222 dramatically reduced the sensitivity of Dex-sensitive HMCLs to Dex,while the inhibitions of mi R-221/222 partially overcame the resistance of Dex-resistant HMCLs to Dex.Conclusion: These data indicate that the expressions of mi R-221/222 are dysregulated in MM cells,which may contribute to the Dex resistance of MM.Part 2.Dexamethasone induces pro-death autophagy in multiple myelomaObjective: To investigate whether autophagy is involved in Dex-induced MM cell death.Methods: To detect the change of autophagy in MM cells treated with Dex,transmission electron microscopy was performed to detect morphological characteristics,western blot analysis was used to detect the expression levels of LC3 B and p62,and confocal microscopy was used to detect the expression of GFP-m Cherry-LC3 fusion protein.MM cells were pretreated with autophagy inhibitor 3-MA or Ly294002 followed by Dex,and then the expression levels of p62 and LC3 B protein were detected by western blot analysis and cell viability was assessed using the CCK-8 assay.Results: Dex induced the occurrence of typical morphological features of autophagosomes and autolysosomes,increased LCB-II:LC3B-I ratio and p62 degradation,and increased number of yellow and red dots in Dex-sensitive cells,but not in Dex-resistant MM cells.What’s more,the addition of 3-MA or LY294002 could inhibit Dex induced autophagy in MM cells,as confirmed by reduced LC3 B conversion and p62 degradation,and significantly protect MM.1S,U266,and JJN3 cells from Dex-induced cell death.Conclusion: These data indicate that the occurrence of autophagy is critical for Dex-induced cell death of MM cells.Part 3.miR-221/222 inhibit autophagy by directly targeting ATG12 and p27-m TOR pathway in multiple myelomaObjective: To investigate the crosstalk between mi R-221/222 and autophagy.Methods: The bioinformatics mi RNA target prediction softwares were used to predict the target genes of mi R-221/222.The dual-luciferase reporter assay was performed to confirm whether the predicted genes were the target genes of mi R-221/222.q RT-PCR and western blooting were performed to analyze the correlation between mi R-221/222 and ATG12 or p27 expressions in CD138+ cells from MM patients and in human MM cell lines.MM cells were transfected with agomir-221/222,antagomir-221/222,ATG12 si RNA or p27 si RNA,and then western blotting was performed to detect the expression of ATG12,P27,pm TOR,m TOR,LC3 B,and p62 proteins and confocal microscopy was used to detect the expression of GFP-m Cherry-LC3 fusion protein.Results: The result showed that ATG12 and p27 were predicted as the potential target genes of mi R-221/222 by bioinformatics mi RNA target prediction softwares and these results were further confirmed by dual-luciferase reporter assay.Correlation analysis showed an inverse correlation between mi R-221/222 and ATG12 or p27 expression in MM patients and human MM cell lines.Furthermore,the expression levels of ATG12 and p27 were upregulated in MM.1S cells compared to MM.1R cells.Transfection of agomir-221/222 inhibited ATG12 and p27 expression at both m RNA and protein levels in MM.1S cells,whereas antagomir-221/222 led to increased expression of ATG12 and p27 in MM.1R cells.Moreover,overexpression of mi R-221/222 reduced LC3B conversion,p62 degradation and increased m TOR phosphorylation in MM.1S cells.Consistently,both the number of yellow and red dots was decreased in MM.1S cell transfected with agomir-221/222.In addition,downregulation of mi R-221/222 led to the opposite effects in MM.1R cells.Moreover,knockdown of ATG12 or p27 decreased the level of autophagy in MM.1S cells and attenuated the effect of antagomir-221/222 on autophagy in MM.1R cells.Conclusion: These data indicate that ATG12 and p27 are the targets of mi R-221/222 and can be regulated by mi R-221/222 in MM cells.mi R-221/222 inhibit autophagy by directly targeting ATG12 and p27 in MM.Part 4.Inhibition of mi R-221/222 induces autophagic cell death in multiple myelomaObjective: To investigate the role of mi R-221/222 mediated inhibition of autophagy in dexamethasone resistance of multiple myeloma.Methods: MM.1S and MM.1R cells were treated with Dex,and then the expression levels of mi R-221/222,ATG12,p27,pm TOR,and p62 were evaluated by q RT-PCR and western blotting.NOD/SCID mice were subcutaneously injected with MM.1S cells or MM.1R cells to establish human Dex-sensitive or Dex-resistant MM xenografts,and then were intraperitoneally treated with Dex,the tumor volume of xenografted mice and the expression levels of mi R-221/222,ATG12,p27,and p62 in tumor tissue were evaluated.MM.1S cells transfected with ATG12 si RNA or p27 si RNA and MM.1R cells co-transfected with antagomir-221/222 and si RNA targeting ATG12 or p27 were treated with Dex,and then apoptosis was examined by detecting cleavage of caspase-3 and PARP using western blot analysis and cell viability was assessed using CCK-8 assay.MM.1R-xenografted mice were treated antagomir-221/222 plus Dex,the tumor volume of xenografted mice and the expression levels of mi R-221/222,ATG12,p27,p62,Beclin-1,and LC3 B in tumor tissue were evaluated.Results: Both the in vitro and in vivo results showed that compared with MM.1R cells,the basal expression levels of mi R-221/222 were lower in Dex-sensitive MM cells.In addition,Dex treatment could further decrease mi R-221/222 expression in Dex-sensitive MM cells,accompanied by upregulated expression of ATG12 and p27,as well as downregulated pm TOR and p62,but these changes were not observed in Dex-treated MM.1R cells.Knockdown of ATG12 or p27 in MM.1S cells inhibited Dex-induced cell death of MM.1S cells,without alteration of Dex-induced cleavage of caspases-3 and PARP.Combined treatment of antagomir-221/222 and Dex could also induce apoptotic cell death of MM.1R cells as indicated by increased cleavage of caspases-3 and PARP,knockdown of p27 or ATG12 had no effect on caspases-3 and PARP.What’s more,pre-treatment with pan-caspase inhibitor z-VAD-fmk completely blocked apoptosis,but only partially inhibit cell death of MM.1R induced by antagomir-221/222 plus Dex.Compared with control groups,combined treatment with antagomir-221/222 and Dex overcame Dex-resistance,as evidenced by a remarkable tumor growth inhibition.In addition,combination treatment induced the activation of ATG12/P27-m TOR autophagy pathway.Conclusion: These data indicate that Dex induces autophagic cell death of MM cells through activation of mi R-221/222-ATG12/p27-m TOR pathway,inhibition of mi R-221/222 to induce pro-death autophagy may be promising therapeutic strategies for the treatment of Dex-resistant MM. |
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