Effect Of Silencing NUPR1 On Autophagy On Human Myeloma Cells And Its Mechanism

Objective Multiple myeloma(MM)is a malignant plasma cell proliferative disease.The regulation of autophagy is closely related to the development of MM.The AKT/mTOR pathway is an important pathway for the regulation of autophagy.Nucleoprotein 1(NUPR1)as a multifunctional small molecule protein can participate in different signal transduction pathways,regulate a variety of tumor cell cycle,apoptosis and autophagy,and thus affect the survival and progression of the tumor.This study will explore the effect and mechanism of silencing NUPR1 on autophagy of human multiple myeloma U266 and RPMI8226 cells,in order to open up new ideas for MM prevention and treatment research.Methods Normal human bone marrow mononuclear cells as control,quantitative real-time PCR(qRT-PCR)was used to detect the mRNA level of NUPR1 and autophagy-related genes(ATG5 and Beclinl)in MM cells.The NUPR1 shRNA lentiviral vector containing the small hairpin RNA(shRNA)targeting NUPR1 gene was constructed and transfected into MM cells.The experiment was divided into parental group,control shRNA-transfected group and NUPR1 shRNA-transfected group.The interference effects of NUPR1 and autophagy related proteins(Beclinl,ATG5,LC3 ?/LC3 ?,P62)and related pathway proteins(p-AKT/T-AKT,p-mTOR/T-mTOR)were detected by Western Blot.Autophagy was measured by transmission electron microscopy(TEM).Results(1)The mRNA expression of NUPR1 and autophagy-related genes(ATG5 and Beclinl)in MM cells is higher than that in normal human bone marrow mononuclear cells.(2)Autophagosomes reduced in NUPR1 shRNA-transfected group under TEM.(3)The expression of NUPR1,ATG5,Beclinl and LC3?/LC3 ? at protein level in NUPR1 shRNA-transfected group was significantly lower than that in parental group and control shRNA-transfected group,while the expression of P62,p-AKT/T-AKT and p-mTOR/T-mTOR is the opposite.(4)After treatment with rapamycin(inhibitor of mTOR)for 24 hours,the expression of autophagy-related proteins(ATG5,Beclinl,and LC3II/LC3I)increased in each group,while the expression of P62,p-AKT/T-AKT and p-mTOR/T-mTOR decreased.Conclusion:Silencing NUPR1 can down-regulate the autophagy of U266 and RPMI8226 cells.Its mechanism may be achieved by regulating the AKT/mTOR pathway.