目的:本研究的目的是检查葡萄糖剥夺对编码糖皮质激素受体(NR3C1)和一些相关蛋白(NR3C2,AHR,NRIP1,NNT,ARHGAP35,SGK1和SGK3)在U87胶质瘤细胞中响应于ERN1/IRE1(内质网到核信号1/肌醇要求酶1)介导的内质网应激信号抑制,以评估其在控制中的可能意义通过IRE1和葡萄糖剥夺介导的内质网应激信号传导来生长胶质瘤。
方法:NR3C1、NR3C2、AHR、NRIP1,NNT,通过实时定量聚合酶链反应研究了空载体pcDNA3.1转染的U87胶质瘤细胞(对照细胞)和无ERN1信号酶功能的细胞(dnERN1转染)在葡萄糖剥夺下的ARHGAP35、SGK1和SGK3基因。
结果:显示NR3C2,AHR,与用葡萄糖生长的对照细胞相比,在葡萄糖剥夺条件下的对照U87神经胶质瘤细胞中SGK1、SGK3和NNT基因上调。同时,NRIP1基因在这些神经胶质瘤细胞中的表达在葡萄糖剥夺下下调,但是NR3C1和ARHGAP35基因对该实验条件具有抗性。我们还表明,ERN1信号酶功能的抑制显着改变了大多数研究基因表达对葡萄糖剥夺条件的反应。因此,葡萄糖剥夺对NR3C2,AHR表达水平的影响,由于NNT基因的表达对葡萄糖剥夺条件具有抗性,因此在ERN1敲低的U87胶质瘤细胞中SGK1基因明显更强。此外,在葡萄糖剥夺条件下,U87胶质瘤细胞中ERN1酶活性的抑制导致ARHGAP35基因表达上调,SGK3基因表达显著下调。
结论:这项研究的结果表明,葡萄糖剥夺不改变NR3C1基因的表达水平,但它显着影响NR3C2,AHR的表达,NRIP,SGK1,SGK3和NNT基因以基因特异性方式在载体转染的U87神经胶质瘤细胞中,可能有助于控制神经胶质瘤的生长,因为大多数研究基因在葡萄糖剥夺条件下的表达显着依赖于IRE1信号传导酶的功能活性。
OBJECTIVE: The aim of the present study was to examine the effect of glucose deprivation on the expression of genes encoded glucocorticoid receptor (NR3C1) and some related proteins (NR3C2, AHR, NRIP1, NNT, ARHGAP35, SGK1, and SGK3) in U87 glioma cells in response to inhibition of endoplasmic reticulum stress signaling mediated by ERN1/IRE1 (endoplasmic reticulum to nucleus signaling 1/inositol requiring enzyme 1) for evaluation of their possible significance in the control of glioma growth through endoplasmic reticulum stress signaling mediated by IRE1 and glucose deprivation.
METHODS: The expression of NR3C1, NR3C2, AHR, NRIP1, NNT, ARHGAP35, SGK1, and SGK3 genes in U87 glioma cells transfected by empty vector pcDNA3.1 (control cells) and cells without ERN1 signaling enzyme function (transfected by dnERN1) under glucose deprivation was studied by real time quantitative polymerase chain reaction.
RESULTS: It was shown that the expression level of NR3C2, AHR, SGK1, SGK3, and NNT genes was up-regulated in control U87 glioma cells under glucose deprivation condition in comparison with the control cells growing with glucose. At the same time, the expression of NRIP1 gene is down-regulated in these glioma cells under glucose deprivation, but NR3C1 and ARHGAP35 genes was resistant to this experimental condition. We also showed that inhibition of ERN1 signaling enzyme function significantly modified the response of most studied gene expressions to glucose deprivation condition. Thus, effect of glucose deprivation on the expression level of NR3C2, AHR, and SGK1 genes was significantly stronger in ERN1 knockdown U87 glioma cells since the expression of NNT gene was resistant to glucose deprivation condition. Moreover, the inhibition of ERN1 enzymatic activities in U87 glioma cells led to up-regulation of ARHGAP35 gene expression and significant down-regulation of the expression of SGK3 gene in response to glucose deprivation condition.
CONCLUSIONS: Results of this study demonstrated that glucose deprivation did not change the expression level of NR3C1 gene but it significantly affected the expression of NR3C2, AHR, NRIP, SGK1, SGK3, and NNT genes in vector-transfected U87 glioma cells in gene specific manner and possibly contributed to the control of glioma growth since the expression of most studied genes in glucose deprivation condition was significantly dependent on the functional activity of IRE1 signaling enzyme.