沉默NUPR1基因对人多发性骨髓瘤U266细胞自噬的影响及机制研究

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

目的:探讨沉默核蛋白1(nuclear protein 1,NUPR1)对人多发性骨髓瘤U266细胞自噬的影响及可能机制。方法:以正常人骨髓单个核细胞为对照,采用实时荧光定量PCR(quantitative real-time PCR,qRT-PCR)检测人多发性骨髓瘤U266细胞NUPR1和自噬相关基因(ATG5和Beclin1)mRNA水平。构建含有靶向抑制NUPR1基因表达的小发夹RNA(small hairpin RNA,shRNA)序列的NUPR1-shRNA慢病毒载体,并将其转染人多发性骨髓瘤U266细胞。实验分为未转染组、阴性对照转染组和转染组,qRT-PCR、Western Blot检测NUPR1干扰效果、3组细胞自噬相关指标(ATG5、Beclin1、P62、LC3Ⅱ/LC3Ⅰ)及相关通路蛋白(p-AKT/T-AKT、p-mTOR/T-mTOR)的表达;单丹磺酰戊二酸(monodansylcadaverine,MDC)染色后,荧光显微镜下观察自噬囊泡的聚集。结果:人多发性骨髓瘤U266细胞较正常人骨髓单个核细胞及转染组细胞NUPR1(F=7.747,P=0.004)、Beclin1(F=5.548,P=0.013)和ATG5(F=4.031,P=0.034)mRNA水平明显增高。转染组NUPR1(F=9.798,P=0.013)、ATG5(F=7.017,P=0.027)、Beclin1(F=7.213,P=0.025)和LC3Ⅱ/LC3Ⅰ(F=7.040,P=0.027)蛋白表达较未转染组和阴性对照转染组明显降低,而P62(F=52.622,P=0.000)、p-AKT/T-AKT(F=6.584,P=0.031)和p-mTOR/T-mTOR(F=7.950,P=0.021)蛋白表达明显增高;转染组细胞胞质内自噬囊泡聚集明显减少(F=12.172,P=0.008)。结论:人多发性骨髓瘤U266细胞NUPR1高表达,自噬水平较正常人骨髓单个核细胞高。沉默NUPR1可下调U266细胞自噬水平。NUPR1基因可能通过AKT/mTOR通路调节U266细胞自噬。

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-transfected 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 measured by monodansylcadaverine(MDC)under fluorescence microscope. Results:The m RNA level of NUPR1(F=7.747,P=0.004),Beclin1(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-transfected 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.