The Clinical And Experimental Study Of NIX In Regulating Of NF-κB In Glioma

Background and ObjectivesAs the most common intracranial tumor,malignant glioma accounts for70%of all primary tumors of the central nervous system.Based on WHO2007Classification: Ⅰ/Ⅱ grade tumors are defined as low-grade glioma, consisted of astrocytoma(AS), pilocytic astrocytoma, etc.;Ⅲ/Ⅳ grade tumors are defined as high-grade glioma,also known as malignant glioma that includes anaplastic astrocytoma(AA), glioblastoma(GBM), anaplastic oligodendroglioma(AOA). In recent years,though the treatment of multi-mode,such as microsurgery, radiotherapy, chemotherapy, and biological therapy,has been adopted, the prognosis of most of the patients are still very pessimistic. The median survival time of patients with newly diagnosed GBM is also time-limited despite of postoperative chemotherapy concurrent with radiotherapy. The results from current clinical follow-up show some patients with malignant glioma still have a longer survival period.There are many questions for the molecular mechanism of resistance to chemoradiotherapy and the prognosis of glioma.To identify differential gene between the tissue of different pathological grade is an effective strategy to elucidate the molecular mechanism that determine the prognosis.Bnip3L, a BH3-only member of the Bcl-2family also known as Nix, is induced by hypoxia, interacts with Bcl-2and Bcl-xl, and acts as a pro-apoptotic factor through its C-terminal transmembrane domain. Nix expression induced by hypoxia retards cancer cell growth. The severity of tumor hypoxia strongly correlates with malignancy and WHO grade.Previous reports implied that Bnip3L and Nix play important roles in hypoxia-dependent cell death and act as tumor suppressors.Nuclear transcription factor kappa B (NF-κB) is an important activator of transcription start, and bind to gene promoter to start the gene transcription in a wide variety of eukaryotic cells. NF-κB is closely related to tumorigenesis and progression of tumor. NF-κB can not only promote tumor development but also inhibit tumor development in specific pathophysiological state, therefore it is necessary to investigate the role of NF-κB in tumors.In this study, the Nix knockdown U251stable cell line was established and used to detect the expression of NF-κB pathway-related proteins. Several experiments in vitro were conducted to observe the cellular behavior under conditions of hypoxia and normoxia. In addition,18cases of astrocytoma (AS),12cases of anaplastic astrocytoma (AA), and16cases of glioblastoma (GBM) were taken to do related detections. To validate data from cells, the clinical outcomes of glioma patients were also reviewed and analyzed.Contents and methods1. The expression of Nix protein and p-NF-kappa B in gliomas and the association between the expression of Nix protein and prognosis.Except for26cases excluded from a total72glioma patients because of death during the perioperative period or due to unavailability of tumor samples,46tumor specimens were obtained and approved pathologically between January2007and January2009, including18cases of astroglioma (WHO Grade Ⅱ),12cases of anaplastic glioma (AA, Grade Ⅲ), and16cases of GBM (Grade Ⅳ). All the tissue samples were harvested by physicians from the tumor center. Patients with chemotherapy or radiotherapy before surgery were not recruited for this study. After surgical resection, the tumor tissues were immediately frozen and stored in liquid nitrogen until processing. Following the tumor resection,28patients with high-grade gliomas (Grades Ⅲ and Ⅳ)received radiotherapy plus the concomitant and adjuvant temozolomide treatment.The mRNA levels of Nix and the protein levels of Nix、IκBα and total p-NF-κB respectively were measured by RT-PCR and Western blot in glioma tissues. We use immunohistochemistry to test the expression of Ki-67in46tumor specimens. The percentage of the Ki-67positive region was determined by an average of10fields with high magnification (x400) with the software package Olysia Bioreport (Olympus Company, Japan) to analyze the relationship between the expression of Ki-67protein and Nix in tumor specimens with different pathological type. We use TUNEL assay to analyze the apoptosis of42cases of tumor (four cases bad for the specimen volume and quality could not be detected) and use the Olympus OlysiaBioreport image analysis system to test the apoptotic percentage in the area of10different vision. The relationship between the rate of apoptosis and the expression of Nix protein in tumor tissues with different types of pathology. Kaplan-Meier survival analysis was performed.2. The function of Nix gene and the preliminary molecular mechanism in gliomaSiRNA targeting Nix was transfected into U251glioma cell line in vitro, with si-Scrambled treated as a negative control group (Nix-wt). After48h of transfection,the Nix-kn group as well as the Nix-wt group was divided divided into two which are cultured with hypoxic culture (5%CO2+95%N2) and normal condition respectively. Following12h of hypoxic culture, total proteins and nuclear proteins were extracted for western blot analysis, including nuclear proteins for Electrophoretic Mobility Shift Assay (EMSA). The cells were incubated in LB medium (10g/L tryptone,5g/L yeast extract and10g/L NaCl, pH7.5) followed by fresh DMEM+10%FBS medium, then detected by DAPI dye and flow cytometry (PI/annexin V dyeing) to evaluate cell viability.All data are presented as means±SD. Two independent samples t tests were used to compare the NIX mRNA expression and the Ki-67index in tumor samples. The2independent samples nonparametric test was used to analyze the KPS scores. The Log-rank Test was used to evaluate statistical significance of tumor-free survival. All statistics were done with SPSS15.0software. The statistical significance was defined as a p value<0.05.Results 1.Nix protein positively regulates NF-κB pathway activation in vitroThe protein expression levels of NF-κB, p-NF-κB, IκBα, p-IκBa, IKKa, and p-IKKa were tested in Nix knockdown (Nix-kn) and control (Nix-wt) U251glioblastoma cell lines under conditions of normoxia and hypoxia. When the cells were incubated in DMEM medium under hypoxic conditions, higher levels of Nix p-NF-κB/p65, p-IκBα, p-IKKa, and lower levels of IκBα were detected by western blot in Nix-wt U251cells, but not in Nix-kn U251cells. The protein level of p-NF-κB in nuclei was also determined. The results showed a higher level of p-NF-κB in the Nix-wt U251cells and nuclei subjected to hypoxia but not in the Nix-kn U251cells. The activation of NF-κB was detected using an EMSA and luciferase reporter gene assay.The results showed that hypoxia activated NF-κB in the Nix-wt U251cells but not in the Nix-kn U251cells. These data revealed that Nix protein acts as an important activator of the NF-κB pathway and contributes to cell recovery under oxygen-poor conditions.After Nix-wt and Nix-kn U251cells were incubated in LB medium instead of the DMEM medium for12h, apoptosis was tested using flow cytometry. After LB medium was replaced with fresh DMEM+10%FBS medium, more apoptotic cells were detected in LB Nix-kn cells than in LB Nix-wt cells, with apoptosis percentages of LB Nix-kn and LB Nix-wt U251cells of11.24±2.15%and7.15±0.87%, respectively, compared with apoptosis in corresponding controls of4.04±0.89%and3.09±1.65%, respectively (Figure2A). DAPI dye also showed more dead cells in Nix-kn U251cells incubated in LB. Five hundred cells were randomly selected for analysis,and the rate of cell death for LB Nix-kn and LB Nix-wt U251cells were13.33±3.28%and7.37±1.17%, and for the corresponding controls (DMEM cultured) were3.52±2.4%and3.43±1.47%, respectively. Significant statistic difference was identified in the LB Nix-kn cells comparing to other groups by apoptosis (p=0.037) and death cells (p=0.041) detection, which also indicated that Nix-kn U251cells have a more difficult time recovering than Nix-wt U251cells do.2.The role of Nix and p-NF-κB expression in clinical glioma samplesNix protein level was evaluated by western blot in46glioma samples. Nix protein was more highly expressed (the ratio of Nix/Gapdh>1) in17cases, referred to as Nix (+), including6cases of AS (6/18,33.3%),5of AA (5/12,41.7%),-and6of GBM (6/16,37.5%). By qRT-PCR detection, Nix mRNA was expressed at significantly higher levels in the GBM samples than in the AS and AA samples (p=0.003and0.027,respectively; but no difference was identified between AS and AA samples. When the samples were split into Nix (+) and Nix (-) subgroups, the significant difference in NIX mRNA levels between Nix (+) and Nix (-) was only seen in AA (p=0.04), and not in AA and GBM samples. The asynchronous expression of mRNA and protein suggested that posttranscriptional regulation most likely affected the Nix protein levels in malignant and high-grade gliomas (AA and GBM).In addition, the high expression of Nix protein always accompanied high expression of p-NF-KB (NF-κB activation) in glioma samples of various pathologies. According to the above data for glioblastoma cell lines and clinical samples, the expression of Nix protein probably functions by activating the NF-κB pathway in the tumorigenesis of gliomas.3.Correlation between Nix protein expression and patient prognosisIn order to validate the role of Nix protein during tumorigenesis, the clinical data from glioma patients were reviewed and analyzed. The results of the Ki-67index revealed that there was a significant difference between Nix (+) and Nix (-) in AS samples (p=0.037), but not in AA and GBM samples, which indicated a different tumor invasive behavior in AS. The clinical analysis also indicated better prognosis for Nix (+) AS cases, suggesting a tumor suppressor role of the NIX gene only in AS.Moreover, the apoptosis analysis by TUNEL assay was used to further evaluate the biological function of Nix protein in42cases of clinical glioma samples. Between Nix (+) and (-) groups,significant different apoptosis percentage was only identified in GBM. Interestingly, more severe tissue apoptotic performance happened in Nix (-) samples rather than (+), which indicated contradict function to the pro-apoptotic factor of Nix protein (p=0.015). However, no such difference was identified in AS and AA groups, although the same tendency could be found in AA.Tumor recurrence and the Karnofsky Performance scale (KPS) score were also determined. After statistical evaluation of the KPS scores, a significant difference was found between Nix (+) and (-) groups for AS and AA (p=0.045and0.047). Survival of the Nix (+) and Nix (-) groups of patients were determined by Kaplan-Meier analysis. The tumor-free survival curve consistently showed better prognosis for Nix (+) patients. However, if subdivided into tumors by grade, only in AS Nix (+) was the survival rate better than for Nix (-) patients; this was not true for AA or GBM.ConclusionAnalyzing all of the above data, we proposed that it is most likely that Nix protein plays both positive and negative roles in the tumorigenesis of gliomas. In low-grade gliomas (Grade Ⅱ) with relatively low expression of NF-κB, the cell death-inducing function might predominant, acting as a tumor suppressor. While in malignant gliomas (Grades Ⅲ and Ⅳ),higher expression of the NIX gene and activity of the NF-κB pathway might promote the oncogene function. Furthermore, although different from the protein levels, there was no significant difference in NIX mRNA levels between the Nix (+) and Nix (-) samples in AA and GBM patients. The asynchronous levels of protein and mRNA expression suggested that posttranscriptional regulation of the NIX gene alters the Nix protein level, which should be further investigated in the future.