Effect of silencing NUPR1 gene on autophagy in human multiple myeloma U266 cells and its mechanism

Abstract:

Objective：To investigate the effect of silencing NUPR1 on autophagy in human multiple myeloma（MM） U266 cells and its possible mechanism. Methods：Normal human bone marrow mononuclear cells were taken as control，and quantitative real-time PCR（qRT-PCR） was used to detect the mRNA level of NUPR1 and autophagy-related genes（ATG5 and Beclin1） in MM U266 cells. The NUPR1 shRNA lentiviral vector containing the small hairpin RNA（shRNA） targeting NUPR1 gene was constructed and transfected into MM U266 cells. The experiment was divided into parental group，control shRNA-transfected group and NUPR1 shRNA-trans－fected group. The interference effects of NUPR1，the expression of autophagy related genes（ATG5，Beclin1，P62，LC3Ⅱ/LC3Ⅰ） and related pathway proteins（p-AKT/T-AKT，p-mTOR/T-mTOR） were detected by qRT-PCR and Western blot. Autophagy was mea－sured by monodansylcadaverine（MDC） under fluorescence microscope. Results：The mRNA level of NUPR1（F=7.747，P=0.004），Be－clin1（F=5.548，P=0.013） and ATG5（F=4.031，P=0.034） in MM U266 cells were significantly higher than those in normal human bone marrow mononuclear cells and NUPR1 shRNA-transfected group cells. The expression of NUPR1（F=9.798，P=0.013），ATG5 （F=7.017，P=0.027），Beclin1（F=7.213，P=0.025） and LC3Ⅱ/LC3Ⅰ（F=7.040，P=0.027） at protein level in NUPR1 shRNA-trans－fected group was significantly lower than that in parental group and control shRNA-transfected group，while the expression of P62 （F=52.622，P=0.000），p-AKT/T-AKT（F=6.584，P=0.031） and p-mTOR/T-mTOR（F=7.950，P=0.021） was the opposite. Moreover，the downregulation of NUPR1 suppressed autophagy in U266 cells（F=12.172，P=0.008）. Conclusion：The expression of NUPR1 in MM U266 cells is higher than that in normal human bone marrow mononuclear cells. Silencing NUPR1 can down-regulate the autophagy of U266 cells. The NUPR1 gene may regulate autophagy in U266 cells through the AKT/mTOR signaling pathway.