Effect Of NUPR1 Gene On Autophagy And Apoptosis On Multiple Myeloma And Its Mechanism

ObjectiveThe study aims to investigate the role of NUPR1 in clinical multiple myeloma(MM)patients and MM cells,with focus on cell autophagy and apoptosis,and its possible mechanism to provide a potentially therapeutic target for MM.Methods1.A total of 28 bone marrow samples and the clinical characteristics were collected from newly diagnosed MM patients,and 10 specimens from healthy donors were obtained and designated as normal control.The total RNAs were isolated from mononuclear cells extracted from the samples,and the relative mRNA levels of NUPR1 and autophagic indicators were tested by qRT-PCR.The association between NUPR1 expression,the autophagic indicators in the bone marrow and the clinical characteristics of the patients was analyzed.2.The shRNA lentivirus vector targeting NUPR1 gene(NUPR1-LV)and negative control lentiviral vector(NC-LV)were transfected into humanMM cell lines(U266 and RPMI 8226).The experiments were set up in three groups: Blank(the untransfected group),NC-LV,and NUPR1-LV groups.The transfection efficiency was detected by flow cytometry.The silencing effect of NUPR1 expression was evaluated by qRT-PCR and Western blot.Transmission electron microscopy(TEM)and MDC staining were used to detect the effect of NUPR1 silencing on autophagy.Flow cytometry and Hoechst staining were applied to assess cell apoptosis of each group.Western blot was used to evaluate the expressions of related proteins in autophagy,apoptosis,and PI3K/AKT/mTOR pathway in MM cells before and after autophagy intervention or PI3 K inhibition.Results1.NUPR1 and autophagy indicators(Beclin1,ATG5 and ATG12)were highly expressed in the bone marrow of newly diagnosed MM patients,and NUPR1 overexpression was correlated with staging(both by Revised International Staging System and Durie-Salmon Staging System),levels of hemoglobin and calcium and bone marrow plasma cells ratio of the MM patients.In addition,the NUPR1 level was positively correlated with Beclin1,ATG5,and ATG12 expressions in the specimen of the patients.2.Lentivirus-mediated NUPR1 silence in MM cells.The transfection rates of both NC-LV and NUPR1-LV groups were more than 80%;the NUPR1 silencing group showed significant reduction in the mRNA andprotein levels of NUPR1.3.NUPR1 silencing reduced autophagy activity in MM cells.TEM showed that the number of autophagosomes in the NUPR1-LV group was significantly less than those in the Blank and NC-LV groups,and MDC staining showed a decrease in autophagy punctas in the NUPR1-LV group.Moreover,autophagy related LC3 conversion and Beclin1 expression were downregulated,and P62 was accumulated in MM cells upon NUPR1 silencing.4.NUPR1 silencing induced apoptosis in MM cells.The flow cytometry data showed that the apoptosis rate was higher in NUPR1-LV group compared with NC-LV group.Hoechst staining also presented increased apoptotic cells in the NUPR1 silencing group.Western blot showed that cleaved caspase3 and BAX expressions were upregulated,whereas antiapoptotic BCL2 was downregulated in the NUPR1-LV group.5.NUPR1 silencing induced autophagy-mediated apoptosis.Treatment with rapamycin,an autophagy inducer,inhibited cell apoptosis in the NUPR1 silencing group;while autophagy inhibition by3-methyladenine promoted cell apoptosis upon NUPR1 silencing.6.PI3K/AKT/mTOR signaling activation was involved in autophagy and cell apoptosis upon NUPR1 silencing.Analysis of the mechanism indicated that there was no significant change in total PI3 K,AKT,and mTOR expressions in the NUPR1-LV group,while phosphorylated PI3 K,AKT,and mTOR expressions were increased;treatment with the PI3 K inhibitor,LY294002,increased autophagy activity and decreased apoptosis after NUPR1 knockdown.ConclusionNUPR1 is overexpressed in the bone marrow of MM patients and is highly associated with the clinical characteristics of MM.NUPR1 silencing has significant antitumor effects on MM,induced partly by autophagy-mediated apoptosis via the PI3K/AKT/mTOR pathway.Therefore,NUPR1-targeted therapy may provide a powerful theoretical basis for curing MM.