| Glioma is the most frequent and aggressive primary brain tumor that exhibits malignant aggressiveness as well as poor prognosis.Currently,despite significant progress made in neurosurgery,radiotherapy as well as chemotherapy,the median survival of glioma patients is less than 1 year and the 5-year survival rate is below 5 %.Also,the underlying molecular mechanism of glioma has not been fully identified yet.Therefore,a better understanding of the processes and pathogenesis mechanisms involved in glioma at systemic,cellular,and molecular levels will help identify new diagnostic and therapeutic targets.Nowadays,along with the advance in the research on the molecular etiology and cellular immunology of glioma,novel anticancer strategies that focus on immunological therapy as well as genetic treatment may provide promising breakthroughs for glioma treatment.Inflammation and immune dysfunction are widely regarded to be participated in promoting the development and progression of cancer.An increased level of pro-inflammatory cytokines,such as interleukins(IL),was observed in a variety of solid tumors.In recent years,interleukin-13(IL-13)has received much attention owing to the finding that it is associated with different malignancies and inflammatory diseases,such as autoimmune diseases,pulmonary asthma as well as ulcerative colitis.Moreover,IL-13 plays a crucial role in chemotaxis and migration of lymphocytes,monocytes and fibroblasts.IL-13 also mediates cancer cell proliferation,metastasis as well as angiogenesis,and is involved in the pathogenesis of various cancers.A higher expression of IL-13 is found in colorectal carcinoma,ovarian carcinoma,lung carcinoma,malignant glioma,pancreatic carcinoma,along with AIDS-associated Kaposi's sarcoma.By contrast,most normal cells such as immune cells or endothelial cells express very low level or undetectable expression of IL-13.It has been suggested that IL-13 is responsible for metastasis in ovarian cancer cells by enhancing activator protein 1(AP-1),extracellular signal-regulated kinases 1 and 2(ERK1/2)as well as matrix metalloproteinase(MMP)activities though interleukin-13 receptor subunit alpha-2(IL-13R?2).Furthermore,IL-13R?2 is involved in signal transduction,triggering the activation of various downstream signaling proteins,such as transforming growth factor-?1,PI3 K,ERK/AP-1,Akt and Src.These findings suggested that IL-13 may be a novel therapeutic target for tumor growth and invasion.The Cys-Cys-Cys-His(CCCH)tandem zinc finger protein TTP is a well-characterized RNA-binding protein that is involved in post-transcriptional regulation of many pro-inflammatory cytokine and growth factor,such as IL-6,IL-33,nuclear factor ?B(NF-?B)and HIF-?.TTP promotes rapid mRNA degradation through the ARE motifs present in the 3' untranslated region(3'UTR)of the targeted mRNAs.The mRNAs encoding TNF-?,IL-23 and GM-CSF are stabilized in TTP-deficient mice and in cells derived from these deficient mice.Overproduction of these cytokines in TTP deficient mice results in a severe systemic inflammatory response including arthritis,autoimmunity and myeloid hyperplasia.Collectively,all evidence indicates that TTP is a critical RNA-binding protein in controlling inflammation and maintaining homeostasis.Altered TTP expression may influence the onset and severity of inflammatory syndromes in humans,such as rheumatoid arthritis,systemic lupus erythematosus and ulcerative colitis.In addition to its function in immune response,TTP is also involved in many different pathological and physiological processes,including cell differentiation,apoptosis,and tumorigenesis.TTP binds and destabilizes the m RNAs encoding proto-oncogenes such as tumor necrosis factor-?(TNF-?),vascular endothelial growth factor(VEGF),c-MYC as well as granulocyte monocyte colony stimulating factor.A recent study showed that the level of TTP was down-regulated in a variety of human solid tumor tissues and cell lines,such as colorectal carcinoma,breast carcinoma along with gastric carcinoma.However,whether the dysregulation of TTP contributes to glioma growth and invasion is currently unclear.In this study,the decreased expression of TTP was found in glioma tissues,and inversely correlated with the level of IL-13.Additionally,TTP was found served as a tumor-inhibitor in the growth,migration and invasion of glioma cells through regulation of IL-13.Thus,our study proposed that the potential application of TTP for treating IL-13-regluated tumor promotion.Our research will be discussed from the following three parts.1.Detect the expression of TTP and IL-13 in 50 patients with glioma and the correlation between TTP and IL-13.Objective: To explore the levels of TTP and IL-13 in 50 cases of human glioma specimens,and analyzing the relationship between TTP and IL-13.Methods: A total of 50 primary glioma tissues and 11 normal brain tissues were collected at the Department of Neurosurgery of the First Affiliated Hospital of Wenzhou Medical University,during 2011 to 2012.These patients included 24 women and 26 men,with median age of 64.All the tissues were collected and frozen in guanidinium thiocyanate solution at-80°C for future experiments.The informed consent was provided by all the patients and all the experiments were approved by the Instiute Research Ethics Committee according to the First Affiliated Hospital of Wenzhou Medical University.The expression of TTP and IL-13 in all the human glioma tissues and normal brain tissues were detected by qRT-PCR.The relationship between TTP and IL-13 was analyzed by Spearman correlation test.Results:Our results showed that the glioma tissues had a significantly decreased TTP mRNA and increased IL-13 mRNA levels in 50 patients with glioma comparied with normal brain tissues.The level of TTP was found down-regulated in high grade glioma specimens than that in low grade glioma tissues,and the abundance of TTP was inversely correlated with that of IL-13 in glioma specimens.Conclusions:(1)The mRNA levels of TTP and IL-13 were found to be down-regulated and up-regulated,respectively in glioma specimens,and the expression of TTP was inversely correlated with IL-13 in glioma patients.(2)The expression of TTP was lower in high grade glioma specimens than that in low grade glioma tissues.2.TTP inhibits in vitro glioma cell growth and invasion.Objective: The recombined lentiviruses LV-TTP and LV-shTTP were constructed in SF295 and U251 cells respectively,and investigate the biological functions of TTP on the cell growth,migration and invasion of glioma cells in vitro.Methods: Western blot was used to determined the relative level of TTP in a panel of glioma cell lines,namely SF295,SHG-44,U87 as well as U251,along with NHA,which is a normal human astrocytes.To establish U251 stable cell line transfected with TTP overexpression Lentiviral vector and SF295 stable cell line trasnfected with TTP shRNA Lentiviral vector.Firstly,the expression of TTP was identified by western blot.Secondly,CCK-8 assay was used to explored the proliferation of glioma cells in vitro.Subsequently,the migratory and invasive abilities of U251/TPP cells were evaluated by matrigel counting assay.Results:Our results demonstrated that the expression of TTP was decreased in all the four glioma cell lines than its expression in NHA.As the level of TTP in SF295 cells was significantly up-regulated in contrast to SHG-44,U87 as well as U251,and the level of TTP in U251 cell was significantly lower than that of three other glioma cell lines,SF295 and U251 cells were chosen for the subsequent experiments.(1)The infection of LV-shTTP in SF295 cells led to a remarkable decrease in its expression,whereas the level of TTP was significantly increased after the U251 cells were infected with LV-TTP.(2)The down-regulation of TTP markedly accelerated the cell growth of SF295 cells.By contrast,U251 cells infected with the TPP caused a significant decrease in cell growth compared with the negative control.Next,the results of cell Matrigel invasion assays revealed that the invasive abilities of U251/TPP cells were suppressed.Additionally,compared with control cells,the silencing of TPP by shTPP dramatically boosted the invasion of SF295 cells.Conclusions:(1)The TTP over-expression and shRNA expression vectors were successfully constructed.(2)Our results suggest that the over-expression of TTP may decreases glioma cells growth and invasion in vitro,whereas the knockdown of TTP accelerated the proliferation and invasion of human glioma cells in vitro.3.Effect of IL-13 and its downstream factors on the proliferation and invasion of glioma cells by TTP in vitro.Objective: To explore the mechanisms of TTP exert as a tumor inhibitory factor on glioma.Methods:(1)The proliferation and invasion of glioma cells were evaluated by CCK-8 and matrigel counting assays,respectively after treated with IL-13 alone or IL-13 plus TTP.(2)The levels of IL-13 and its downstream factors in glioma cells were characterized by western blot.Results:(1)Our results showed that the addition of IL-13 alone could sufficiently promote cell growth and invasion in U251 cells.Additionally,treatment with IL-13 attenuated the suppressive effect of TTP on the cell growth and invasion of TTP-overexpressed U251 cells.(2)Our result indicated that the silencing of TTP in SF295 cells increased the protein expressions of IL-13 as well as p-PI3 K,while the levels of IL-13 and p-PI3 K were decreased owing to the effect of TTP infection in U251 cells.Moreover,the level of IL-13 mRNA was decreased and increased respectively after being infected with TTP or TTP shRNA.(3)The levels of p-PI3 K and its downstream target gene p-Akt as well as p-mTOR in TTP-depleted SF295 cells were markedly over-expressed by western blot analysis.Whereas the up-regulation of TTP in U251 cells decreased the phosphorylation of PI3 K,Akt and mTOR.Conclusions:Inconclusion,these observations indicated that the regulation of IL-13/PI3K/Akt/mTOR pathway is associated with the tumorigenicity of TTP in glioma cells.Our findings are consistent with the hypothesis that TTP inhibits tumorigenesis via IL-13/PI3K/Akt/mTOR pathway in glioma,thereby inhibiting cell growth and invasion of glioma cells.In this study,we aimed to investigate the effects of TTP on the proliferation,migration and invasion of human glioma cells as well as the underlying mechanisms,which might benefit the future therapy for malignant gliomas.In this way,promotion of TTP is a potential therapeutic target for glioma prevention and treatment in the future. |
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