Neuroglobin Influences On The Growth Of Mouse Neuroblastoma N2a Cells

Background and ObjectivesNeuroglobin(Ngb), regarded as a specific protein in neurons, now has been detected in various tumors. And it indicates the potential role in tumor genesis, progression and metastasis, besides the adjustment in hypoxia and neuron protection in cerebral ischemia. So far, there are few studies on direct connection between Ngb and tumors genesis. However, the evidence that myoglobin(Mb) and cytoglobin(Cygb), another two members of globin family, are involved in tumor formation has been shown by several institutes. The possible mechanisms involve scavenging reactive nitrogen species(RNS) and reactive oxygen species(ROS), adaptation in hypoxia with regulating expression of hypoxia inducible factor-1(HIF-1), increasing the stability of mitochondrial membrane. All these processes are at least in part associated with the prevention of apoptotic cell death and could be related to a possible interaction between globins and cytochrome c.Considering the previous work that TAT-mediated delivery of Ngb deals with the problem of protecting against focal cerebral ischemia by going through blood-brain barrier(BBB), it is meaningful to explore its potential effect on brain tumor genesis and progression, before the clinical application of Ngb.Here, we intent to explore the expression of Ngb in several cell lines of brain tumor and the effect on tumor genesis and proliferation by the infection of Lenti-Ngb-GFP and the up-regulation of Ngb. It will be the first step before the further study of mechanisms and make it safe for the clinical neural protection therapy in cerebral vascular diseases.Methods 1. Screening for Ngb expression in a neuroblastoma cell lines(N2a) and two astrocytoma cell lines(U251, C6) by reverse transcription-polymerase chain reaction(RT-PCR), immunofluorescence and Western Blot. 2. Lentiviruses were used to up-regulate Ngb expression in N2 a. The outcome was examined by Western Blot. Further experiments were carried out to observe cell proliferation between virus-treated and control groups, including cell viability assay and colony formation assay.Results 1. Ngb expression was found in N2 a as well as astrocytoma cell lines(U251, C6) by RT-PCR, immunofluorescence and Western blotting. 2. After infecting N2 a with Lentiviruses, the expression of Ngb protein was significantly increased in Lenti-Ngb-GFP groups, compared with the control and Lenti-GFP groups. 3. Lenti-Ngb-GFP groups showed a higher ability of cell proliferation especially since the sixth day(P<0.01). Little difference in colony numbers was found between virus-treated and control groups after 14 days cultivation in similar situation. However, the average sizes of colony in Lenti-Ngb-GFP groups were much bigger than in Lenti-GFP and control groups(P<0.05).Conclusion 1. The expression of Ngb in several cell lines of brain tumors has been shown by exerting RT-PCR and immunofluorescence 2. After being infected by Lenti-Ngb-GFP, N2 a was up-regulated the expression of Ngb. 3. In N2 a cell lines the overexpression of Ngb can accelerate the tumor proliferation, which is shown by exerting cell viability and colony formation assay.