LITAF Enhances Radiosensitivity Of Human Glioma Cells Via The FoxO1 Pathway

Background:Gliomas represent 75%of malignant primary brain tumors and are the most common malignant tumors of the central nervous system in adults.Gliomas,based on their growth pattern and the presence or absence of IDH mutations,are categorized into circumscribed gliomas（WHO grade I）and diffusely infiltrating gliomas（WHO grades II-IV;including both astrocytic and oligodendroglial）with the 2016 WHO classification system.Most circumscribed gliomas are benign resectable lesions.Conversely,diffusely infiltrating gliomas are difficult to be cured by resection alone.Among them,more than half are glioblastomas（GBM,WHO grade IV）,which are the most malignant glioma tumors.The annual incidence of glioblastomas is about 3-4 cases per 100,000 individuals^[1-5].At present,maximum surgical resection and adjuvant radiotherapy combined with chemotherapy are the major treatment methods for patients with glioblastomas.Recurrence is the inevitable clinical outcome in most patients with glioma because of the biological characteristics of highly invasive growth and resistance to radiotherapy and chemotherapy of glioma cells.The median survival time is only14.6 months,with death mostly due to recurrence of glioma resulting to cerebral edema or increased intracranial pressure in patients with GBM^[6,7].Therefore,the exploring of molecular mechanisms may help to understand the character of glioma cells in detail and is in favor of looking for a new therapeutic strategies to gliomas.LITAF was originally named PIG7 in 1997 because its expression was able to be induced by P53^[8].Several studies found that LITAF was able to bind to the-550 to-487 promoter region of TNF-αand regulated the expression of TNF-αunder the stimulation of LPS.Then it was named LITAF（LPS-induced tumor necrosis factor alpha factor）which mainly expressed in placenta,peripheral blood leukocytes,lymph nodes,and spleen^[9].The biological functions of LITAF include two aspects:one is to regulate the inflammatory response,and the other is to inhibit tumor growth.In the inflammatory response,LITAF can interact with STAT6（B）and form a complex to activate transcription of inflammatory cytokine genes,such as those encoding TNF-α,IL-6,and CXCL18 in macrophages in response to LPS^[10-12].The expression of LITAF increase in intestinal tissues of patients with inflammatory bowel disease and may be related to the development of Crohn’s disease and ulcerative colitis by activating transcription of genes encoding inflammatory factors^[13].It has also been considered to be correlated with the incidence of psoriasis^[14]and osteoarthritis^[15].In recent years,many studies highlighted LITAF as a tumor suppressor which inhibited cell growth and promoted apoptosis in prostate cancer cells^[16,17],pancreatic cancera^[18],and B-cell lymphoma^[19].In our knowledge,the expression and function of LITAF in human gliomas remain unexplained.Survey of the expression and role of LITAF in gliomas may help promote the new therapeutic targets for gliomas.FoxO1 belongs to the Forkhead box O（FoxO）transcription factor family,which has four subtypes in humans:FoxO1,FoxO3,FoxO4 and FoxO6.The role of FoxO proteins are associated with regulation of oxidative stress,cell differentiation,apoptosis,proliferation and DNA repair^[20].FoxO1 is a major targeted protein in activation of P13K/AKT signaling,which regulated cell proliferation,apoptosis and DNA repair^[21].It is known as a tumor suppressor because of its expression degrading or deleting in various tumors,such as Hodgkin’s lymphoma^[22],breast cancer^[23],alveolar rhabdomyosarcoma^[24]and gliomas^[25].Several studies demonstrated that FoxO1 as pro-apoptotic factor can be activated by TNF-α^[26-29].Then,we hypothesis FoxO1 may play an important role in LITAF pathway to regulate the biological functions of glioma cells.Taken together,the purpose of this study was to investigate the expression of LITAF in glioma tissues and the effect of LITAF on proliferation,apoptosis and radiosensitivity of glioma cells.The role of FoxO1 in LITAF pathway was analyzed at the same time.Methods:1.Expression of LITAF in normal brain tissue and glioma tissues.1.1 The expression of LITAF in glioma tissues and normal brain tissues were analyzed in TCGA database using the online website FireBrowse（http://firebrowse.org/）.1.2 Clinical specimens of surgically resected glioma tissues and normal brain tissues were collected,and the levels of LITAF mRNA and protein expression were detected by qRT-PCR and Western blot.2.The effect of LITAF expression on the prognosis of patients with glioma.Download clinical data of patients with glioma in TCGA database,and analyze the effect of LITAF expression on the OS of patients with glioma using Kaplan Meier.3.The proliferation and apoptosis of glioma cells were detected after knockdown or overexpression of LITAF.3.1 Knockdown or overexpression of LITAF by lentiviral vector transfecting in glioma cells.3.2 The proliferation of glioma cells was surveyed with EdU after knockdown or overexpressing of LITAF gene.3.3 The apoptosis of glioma cells was detected by flow cytometry after knockdown or overexpressing of LITAF gene.4.The radiosensitivity of glioma cells was analyzed after LITAF knockdown or overexpression.4.1 Knockdown or overexpression of LITAF by lentiviral vector transfecting in glioma cells.4.2 Flow cytometry was used to detect the apoptosis of glioma cells received ionizing radiation after LITAF knockdown or overexpression.4.3 Colony formation assay was used to detect colony formation of glioma cells received ionizing radiation after LITAF knockdown or overexpression.5.Expression of FoxO1 and its downstream signaling pathways TRAIL,FASLG,and BIM in glioma cells received ionizing radiation after LITAF knockdown or overexpression.5.1 Stably knockdown or overexpressing LITAF in glioma cells by transfection with lentiviral vector.5.2 The expression of FoxO1 and its downstream signaling pathways TRAIL,FASLG,and BIM in glioma cells received ionizing radiation was detected with qRT-PCR and Western blot.Results:1.Expression of LITAF in glioma tissues.1.1 The levels of LITAF mRNA in glioma tissues increased compared to normal brain tissues in TCGA database.1.2 The levels of LITAF mRNA in glioma tissues collected by us decreased in qRT-PCR analysis compared to non-tumor brain tissues.And the results of Western-blotting also showed lower levels of LITAF protein in glioma tissues compared to non-tumor brain tissues.1.3 Showing in the result of Kaplan-Meier analysis by TCGA database,lower expression of LITAF predicted a better prognosis in patients with glioma received radiotherapy than high expression of LITAF.2.Intervening of LITAF expression did not influence directly the proliferation and apoptosis of glioma cells.2.1 The results of EDU assay suggested that proliferation of glioma cells did not have significant different after knockdown or overexpression of LITAF in glioma cells.2.2 The results of flow cytometry assay demonstrated that overexpression of LITAF had no effect on apoptosis in glioma cells,the same results were observed in glioma cells with LITAF knockdown.3.Expression of LITAF enhances radiosensitivity of glioma cells.3.1 The assessment of apoptosis by flow cytometry showed that knockdown of LITAF gene inhibited the proapoptotic effects of ionizing radiation on glioma cells.In contrast,the rate of apoptotic glioma cells increased markedly in LITAF overexpression groups received ionizing radiation compared to control group.3.2 The results of colony formation assays showed that glioma cells with LITAF knockdown were resistance to ionizing radiation,and apoptosis of glioma cells with LITAF overexpression increased remarkable after ionizing radiation compared to glioma cells of control groups.4.LITAF enhanced radiosensitivity of glioma cells via FoxO1 pathway.The results of qRT-PCR and Western-blot showed that expression of FoxO1 and its downstream targets such as Bim,TRAIL,and FASLG also increased in glioma cells received ionizing radiation along with increasing of LITAF expression.Conclusions:1.Expression of LITAF in glioma tissues was higher in TCGA database than normal brain tissue.In contrast,the level of LITAF expression in glioma tissues was lower in our collected glioma samples than normal brain tissues.2.The results of this study suggested that overexpression of LITAF predicted a poor survival in patients with glioma.3.Intervening of LITAF expression by RNAi or overexpressing did not influence directly the proliferation and apoptosis of glioma cells.4.Expression of LITAF enhanced the radiosensitivity of glioma cells via the FoxO1 pathway.