上调NPRL2表达对神经胶质瘤SHG44和U251细胞增殖的抑制作用

Up-regulation of nitrogen permease regulator like-2 expression inhibits the malignant growth of glioma SHG44 and U251 cells

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 over-expression on the possible mechanism. the effects of nitrogen proliferation of human permease regulator glioma SHG44 and like-2 （NPRI_2） gene U251 cells, and its Methods： After recombinant lentiviral LV-NPRL2 was infected into glioma SHG44 and U251 cells, the expression levels of NPRL2 mRNA and protein were detected by real- time fluorescent quantitative PCR and Western blotting, respectively. The proliferation and cell cycle distribution of SHG44 and U251 cells were tested by CCK-8 method and FCM, respectively. The expression levels of phospho-3-phosphoinositide-dependent protein kinase 1 （p-PDK1）, phospho-protein kinase B1 （p-PKB1, p-Aktl）, p27 and p21 proteins were detected by Western blotting. The enzyme activity of PDK1, Aktl, cyclin-dependent kinase （CDK） 2 and CDK4 was also measured by enzyme-linked immunometric meter. The effect of over-expression of NPRL2 in SHG44 and U251 cells on ~umorigenicit~ of subcutaneous xenografted tumor in nude mice and proliferating cell nuclearantigen （PCNA） and Ki-67 of xenografted tumor tissues were detected by immunohistochemistry. Results： The expression levels of NPRL2 mRNA and protein in SHG44 and U251 cells in LV- NPRL2 infection group were higher than those in the negative control [SHG44 and U251 cells infected with lentivirus negative control （LV-NC）] and blank control （SHG44 and U251 cells without any infection） （all P 〈 0.01）. The cell proliferation of LV-NPRL2 infection group were obviously inhibited （all P 〈 0.05）, the percentage of cells was increased in phase G0/G1 （P 〈 0.01） and decreased in phase S （P 〈 0.01）. The expression levels of p-PDK1 and p-Aktl in SHG44 and U251 cells of LV-NPRL2 infection group were down-regulated, and the expression levels of p21 and p27 proteins were opposite （all P 〈 0.01）, as well as the activities of PDK1, Aktl, CDK2 and CDK4 were decreased （all P 〈 0.01）. The ability of tumorigenesis （P 〈 0.05） and PCNA and Ki-67 in LV-NPRL2 group were lower than those in the negative control group （all P 〈 0.01）. Conclusion： NPRL2 over-expression may inhibit the proliferation of human glioma SHG44 and U251 cells. This effects may be related to the inhibition of PDK1/Aktl signaling pathway.