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目的:探讨抑癌基因NPRL2(nitrogen permease regulator like-2)过表达对人神经胶质瘤SHG44和U251细胞增殖的影响及其可能的作用机制。方法:将NPRL2过表达的重组慢病毒LV-NPRL2感染SHG44和U251细胞后,应用实时荧光定量PCR和蛋白质印迹法检测细胞中NPRL2m RNA和蛋白的表达水平,CCK-8和FCM法检测细胞增殖和细胞周期,蛋白质印迹法检测磷酸化3-磷酸肌醇依赖性蛋白激酶1(phospho-3-phosphoinositide-dependent protein kinase 1,p-PDK1)、磷酸化蛋白激酶B1(phospho-protein kinase B1,p-PKB1,又称p-Akt1)、p27和p21蛋白的表达水平,应用酶联免疫检测仪检测细胞中PDK1、Akt1、细胞周期蛋白依赖性激酶2(cyclin-dependent kinase 2,CDK2)和CDK4的活性。裸鼠皮下成瘤实验观察NPRL2过表达对SHG44和U251细胞致瘤性的影响,免疫组织化学法检测裸鼠移植瘤组织中增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)和Ki-67增殖指数的变化。结果:LV-NPRL2感染组SHG44和U251细胞中NPRL2 m RNA和蛋白的表达水平均显著高于阴性对照组[SHG44和U251细胞感染阴性对照慢病毒(lentivirus-negative control,LV-NC)]和空白对照组(SHG44和U251细胞未感染任何慢病毒)(P值均<0.01)。LV-NPRL2感染组SHG44和U251细胞的增殖受到抑制(P值均<0.05),G0/G1期细胞所占百分比显著上升(P值均<0.01),S期细胞所占百分比显著下降(P值均<0.01),p-PDK1和p-Akt1蛋白的表达水平下调(P值均<0.01),p21和p27蛋白的表达水平上调(P值均<0.01),PDK1、Akt1、CDK2和CDK4的活性水平显著下降(P值均<0.01)。LV-NPRL2感染组SHG44和U251细胞在裸鼠皮下的成瘤能力(P<0.05)以及移植瘤组织中PCNA和Ki-67的增殖指数显著低于阴性对照组(P值均<0.01)。结论:NPRL2过表达可以抑制人神经胶质瘤SHG44和U251细胞的增殖,其机制可能与抑制PDK1/Akt1信号转导通路有关。
Objective: To investigate the effect of tumor suppressor gene NPRL2 (overexpression of nitrogen permease regulator like-2) on the proliferation of human glioma SHG44 and U251 cells and its possible mechanism. Methods: NPRL2mRNA and protein expression were detected by real-time fluorescence quantitative PCR and Western blotting after infected with NPRL2 recombinant lentivirus LV-NPRL2. Cell proliferation and proliferation were detected by CCK-8 and FCM Cell cycle and Western blotting were used to detect phospho-3-phosphoinositide-dependent protein kinase 1 (p-PDK1), phospho-protein kinase B1 (p- PKB1, also known as p-Akt1), p27 and p21 protein levels, the activities of PDK1, Akt1, CDK2 and CDK4 were detected by enzyme-linked immunosorbent assay . The effect of NPRL2 overexpression on tumorigenicity of SHG44 and U251 cells was observed by subcutaneous tumor formation in nude mice. The proliferating cell nuclear antigen (PCNA) and Ki-67 proliferation index in nude mice xenografts were detected by immunohistochemistry Variety. Results: The expression levels of NPRL2 m RNA and protein in SHG44 and U251 cells were significantly higher than those in the negative control group (LV-NC and SHG44 and U251 cells) The control group (SHG44 and U251 cells were not infected with any lentivirus) (P values <0.01). The proliferation of SHG44 and U251 cells was inhibited in LV-NPRL2 infection group (all P <0.05), the percentage of cells in G0 / G1 phase increased significantly (P <0.01), and the percentage of S phase cells decreased significantly (P value (All P <0.01). The expressions of p-PDK1 and p-Akt1 were down-regulated (P <0.01), the expressions of p21 and p27 were up-regulated Level decreased significantly (P <0.01). The tumorigenicity of SHG44 and U251 cells in nude mice (P <0.05) and the proliferation index of PCNA and Ki-67 in LV-NPRL2 infected group were significantly lower than those in negative control group (all P <0.01). CONCLUSION: Overexpression of NPRL2 can inhibit the proliferation of human glioma SHG44 and U251 cells, which may be related to the inhibition of PDK1 / Akt1 signal transduction pathway.