Knockdown of NRAGE induces odontogenic differentiation by activating NF-κB signaling in mouse odontoblast-like cells

Purpose: Neurotrophin receptor-interacting MAGE homologue (Nrage) plays an important role in bone development and the metabolism of normal skeletal structures. Our previous study showed that Nrage inhibited the odontogenic differentiation of mouse dental pulp cells. However, the potential roles and mechanism of Nrage in regulating odontogenic differentiation are unknown. The aim of this study was to investigate the molecular mechanism of Nrage in odontogenic differentiation of mouse odontoblast-like cells.

Materials and methods: Endogenous expression of Nrage was stably downregulated by lentivirus-mediated shRNA. Mineralized nodules formation was detected by alizarin red S staining. Dmp-1, Dspp, and ALP mRNA and protein levels were detected by qRT-PCR and western blotting, respectively. In addition, ALPase activity was detected. Confocal microscopy and co-immunoprecipitation (co-IP) were used to analyze the interactions between NRAGE and NF-κB signaling molecules. An IKK inhibitor was also used in the study.

Results: NRAGE expression in odontoblasts was downregulated during mouse first maxillary molar development. Moreover, NRAGE expression was downregulated during odontogenic differentiation of odontoblast-like cells. NRAGE knockdown significantly upregulated DMP1 and DSP expression, increased ALPase activity, and promoted mineralized nodule formation. In addition, NRAGE knockdown increased the translocation of NF-κB1 to the nucleus and phosphorylation levels of p65. Co-IP results showed that NRAGE bound to IKKβ. Most importantly, the promoting effect of Nrage knockdown on odontoblastic differentiation was reduced after treatment with an IKK inhibitor.

Conclusions: Our data confirmed that NRAGE is an important regulator of odontogenic differentiation of odontoblasts by inhibiting the NF-κB signaling pathway through binding to IKKβ.

Abbreviations: Nrage: neurotrophin receptor-interacting MAGE homologue; DSP: dentin sialophospho protein; DMP-1: dentin matrix protein-1; BMP: bone morphogenetic protein; Wnt: wingless; NF-κB: nuclear factor of activated B cells; DAPI: 4′,6-diamidino-2-phenylindole; KO: knockout; DPCs: dental pulp cells; AA: ascorbic acid; β-Gly: β-glycerophosphate; Dex: dexamethasone; co-IP: co-immunoprecipitation; IκB: inhibitor of NF-κB; IKK: IκB kinase     

NRAGE suppresses metastasis of melanoma and pancreatic cancer in vitro and in vivo

We previously reported that human NRAGE could significantly alter the cellular skeleton and inhibit cell–cell adhesion, suggesting that human NRGAE play a potential role in cellular motility. Here, we report overexpression of human NRAGE in PANC-1 and B16-Bl6 cells could significantly suppress the metastasis of these cells in vitro and in vivo. Consistently, PANC-1 with stable silencing of NRAGE by RNA interference, exhibits a more metastatic phenotype than the native cell. Expression of epithelial proteins, including E-cadherin and β-catenin is down regulated in siRNA-NRAGE PANC-1 cells. Further studies find that overexpression of human NRAGE suppresses the mRNA expression and activity of MMP2 significantly. Summary, our studies indicate for the first time that NRAGE could suppress metastasis of melanoma and pancreatic cancer probably through downregulation of MMP-2.     

Relationship between NRAGE and the radioresistance of esophageal carcinoma cellline TE13R120

Background and Objective: The mRNA levels of 59 genes, detected by cDNA microarray, were up-regulated in the radioresistant human esophageal cacinoma cell line TE13R120 as compared with its parental cell line TE13 before and after radiation, and the expression of NRAGE gene showed a gradually up-regulating tendency. This study aimed to further detect the differences of NRAGE gene and protein expression and apoptosis between TE13R120 and TE13 cells, and to investigate the relationship between the NRAGE and t...     

食管癌细胞系TE13R120放射抗性与HDAC3、NF-kB和NRAGE关系的研究

目的探讨HDAC3、NF—kB和NRAGE基因与食管癌细胞放射抗性的关系。方法以人食管癌细胞系TE13和由TE13经γ射线反复照射建立的放射抗性细胞系TE13R120为研究对象，用Western blotting技术检测这两种细胞于不同时间、剂量点照射后基因HDAC3、NF—kB和NRAGE的蛋白表达；用流式细胞术检测相同时间、剂量点两种细胞的凋亡和细胞周期分布情况。结果Western blotting显示，与TE13相比，TE13R120细胞照射前后HDAC3的核表达均增高，NF-kB的表达也明显增强，NRAGE在两种细胞胞浆中的表达无差异。与TE13相比，TE13R120中S期细胞明显增多，照射后G2期阻滞明显，凋亡水平较低。结论基因HDAC3和NF-kB可能在食管癌细胞放射抗性形成中发挥作用并可能有协同作用；NRAGE与食管癌辐射抗性的关系有待进一步研究。     

NRAGE食管鳞癌表达与放疗疗效关系初探

目的 检测NRAGE蛋白在食管鳞癌中表达,探索NRAGE与放疗疗效之间关系。方法 采用免疫组化S-P法检测44例患者食管鳞癌组织NRAGE表达情况,结合患者临床数据行统计学分析。Cox模型多因素分析。结果 食管鳞癌组织中NRAGE蛋白总体表达水平、核蛋白表达水平均与近期疗效呈负相关(P=0.025、0.008)。NRAGE总体蛋白表达呈强阳性组和阳性+弱阳性组3年生存率分别为16%和36%(P=0.198),NRAGE核蛋白表达呈强阳性组和阳性+弱阳性组3年生存率分别为0%和41%(P<0.001)。多因素分析结果显示NRAGE核表达呈强阳性组比阳性+弱阳性组的死亡风险高(P=0.002)。结论 食管癌组织中NRAGE蛋白总体表达程度与放疗近期疗效呈负相关,与远期生存不相关;NRAGE蛋白核内强阳性表达程度与放疗近期疗效呈负相关,与远期生存呈负相关。提示NRAGE可能是预测食管鳞癌放射抗性乃至放疗疗效的分子指标。     

MAGE-A antigens as targets in tumour therapy

MAGE-A proteins constitute a sub-family of Cancer-Testis Antigens which are expressed mainly, but not exclusively, in germ cells. They are also expressed in various human cancers where they are associated with, and may drive, malignancy. MAGE-A proteins are highly immunogenic and are considered as potential targets for cancer vaccines and/or immuno-therapy. Moreover, recent advances in our understanding of their molecular pathology have revealed interactions that offer potential as therapeutic targets. Here we review recent progress in this area and consider how these interactions might be exploited, especially for the treatment of malignant cancers for which available treatments are inadequate.     

神经生长因子受体介导的黑色素瘤抗原编码基因同源蛋白对牙髓细胞增殖的影响

目的探索神经生长因子受体介导的黑色素瘤抗原编码基因同源蛋白(NRAGE)对人牙髓细胞(h DPCs)和小鼠成牙本质细胞(MDPC-23)细胞增殖的影响。方法重组慢病毒转染细胞稳定敲除h DPCs和MDPC-23的NRAGE表达,体外组织块法原代培养h DPCs和MDPC-23,进而检测NRAGE对h DPCs和MDPC-23的增殖影响。采用CCK-8法分析NRAGE对h DPCs和MDPC-2细胞增殖的影响,流式细胞术分析NRAGE对h DPCs和MDPC-23的细胞周期分布和细胞凋亡影响。免疫荧光法检测NRAGE和NF-κB的表达和定位,分析NF-κB蛋白表达水平,并用IKK抑制剂处理细胞后,分析细胞周期和细胞凋亡。结果重组慢病毒转染后NRAGE的mRNA和蛋白水平下降显着。NRAGE敲减后抑制了h DPCs和MDPC-23的增殖活性和凋亡。NRAGE敲减后显示h DPCs的G0G1期滞留显著,而对MDPC-23没有影响。同时,NRAGE敲减后激活NF-κB信号通路。IKK抑制剂可以抑制NRAGE敲除后对h DPCs和MDPC-23的细胞凋亡的抑制作用。结论 NRAGE敲减后抑制牙髓细胞的增殖活性。NRAGE通过NF-κB信号通路调控h DPCs的细胞周期和凋亡。     

NRAGE基因在胃癌组织中表达与病理特征的关系

目的 用原位杂交方法检测NRAGE基因mRNA在胃癌组织、癌旁组织和正常胃黏膜组织中的表达.方法 cDNA探针的制备,用原位杂交的方法检测36例胃癌组织、16例癌旁组织和11例正常胃黏膜组织中NRAGE基因mRNA的表达状况.结果 在正常胃黏膜组织中NRAGE基因mRNA的表达较胃癌组织和癌旁组织中的表达明显升高(P＜0.05);胃癌组织中NRAGE mRNA的表达,与胃癌的浸润深度、淋巴转移、临床分期密切相关(P＜0.05).结论 胃癌的发生可能与该基因的低表达有关,NRAGE基因可能在胃癌的发生过程中起抑制作用.     

Tumor-specific MAGE proteins as regulators of p53 function

Since its discovery in 1991, the knowledge about the tumor specific melanoma antigen gene (MAGE-I) family has been continuously increasing. Initially, MAGE-I proteins were considered as selective targets for immunotherapy. More recently, emerging data obtained from different cellular mechanisms controlled by MAGE-I proteins suggest a key role in the regulation of important pathways linked to cell proliferation. This is in part due to the ability of some MAGE-I proteins to control the p53 tumor suppressor. In this review, we focus on the mechanisms proposed to explain how MAGE-I proteins affect p53 functions.