The Role Of IRG1 And Its Associated Mechanism In Glioma Proliferation And Clinical Significance

BACKGROUNDGlioma is the tumor in the neural ectoderm, therefore, also called neural ectoderm tumor or neuroepithelial tumor accounted for 40-50% of brain tumors.Most gliomas originated in different types of glial cells, but according to group similar genetic origin and biological characteristics, commonly known as glioma.Glioma classification method are many, all types of glioma with astrocytoma most, glioblastoma, medulloblastoma, ependymoma times [1].Various types of gliomas with different parts, such as glioblastoma almost occurred in the cerebral hemisphere glioblastoma almost all occur in the brain hemisphere;Medulloblastoma in cerebellar vermis;More than ependymoma in fourth ventricle;Tu glioma occurs mostly in less in cerebral hemisphere;Astrocytoma adults see more at the brain hemisphere, children are more in the cerebellum.Glioma in men see more, especially in polymorphism glioblastoma, medulloblastoma, more men than women significantly.Each glioblastoma found in middle age, ependymoma in children and youth, medulloblastoma almost occurred in the children.Glioma of parts and age have some relations, such as the brain astrocytoma and glioblastoma mostly in adults, the cerebellum glioma (astrocytoma, medulloblastoma, ependymoma) found in children [2].Glioma mostly slow onset, since the onset of symptoms to clinical time generally from several weeks to months, a few of years.High malignant degree and posterior fossa tumor history is shorter, a longer history of relatively benign tumor.Tumor if any bleeding or cystic change, will suddenly worse, even a process similar to the pathogenesis of cerebrovascular disease.Glioma clinical symptoms can be divided into two aspects, on the one hand, increased intracranial pressure symptoms, such as headache, vomiting, decreased visual acuity, diplopia, mental symptoms and so on; On the other hand are a result of the tumor oppression, infiltration, damage brain tissue of focal symptoms, early can be characterized by stimulating symptoms such as localized epilepsy, characterized by loss of neurologic symptoms such as paralysis.Inflammatory response refers to the biological tissue is trauma, hemorrhage, or some pathogen infection, stimulate physiological responses.Including the redness and swelling, fever, pain and other symptoms.Inflammatory reaction is the innate immune system to remove harmful stimulus or originate and promote the protection of the repair, not like the acquired immune system for a particular originate [3].Inflammation infection is not synonymous with, even if there is a lot of inflammation is due to infection, inflammation is one of the response of organisms to originate.Typically, inflammation is beneficial, is the body’s automatic defense reaction, but sometimes, inflammation can cause the body’s own immune system, and attacks its own tissue and cells.Inflammation is the body to fight infection, tissue damage, harmful stimulus such as an important defense mechanism, but in many cases, excessive inflammation itself will cause damage to the body.Inflammatory reaction can stimulate neutrophils and macrophages engulf pathogens and necrosis collapse of cell debris, and macrophages in the body’s nonspecific immune function plays an important role.Studies have shown that in the early cancer from the healthy tissue macrophages after activation can inhibit the growth of tumor cells, but in the stage of tumor progression, from the inside of the tumors had the macrophages are to promote the growth of tumor cells.Macrophages in different stages of the tumor plays a different role.The study found that the height of the macrophage infiltration in tumor tissues is associated with the prognosis of patients with tumor.Research has shown that inhibit the growth of tumors had successfully in chemotherapy or radiotherapy after 3 months of tumors in patients with active inflammation in local, infiltrating cells are activated macrophages and astrocytes, CD3+ and CD8+ lymphocytes and subsequent demyelinating change [4].Visible macrophage mediated inflammatory response has an important position in the treatment of gliomas.METHODChapter lfirst section:The different kinds of glioma cells in vitro, QPCR and Western blot test IRG1 expression differences in glioma cells and normal cells. Knockdown IRG1 observed its effects on glioma cell proliferation and migration. By Western blot to test cell cycle factors, EMT related the swallow and the NK-kB pathway factor levels, screening IRG1 mechanisms through which promote the proliferation of glioma cells.Chapter 1 second section:The biological functions of IRG1 in vivo, weassessed the in vivo role of IRG1 by using a xenograft transplantation model. We subcutaneously injected sh-IRG1 and IRG1 lentiviral infection U251 and SHG-44 cells or control cells into nude mice and monitored tumor growth over a 40-day period.Chapter 2:Cytoplasmic IRG1 protein is a prognostic factor for postsurgicalglioma patients. The expression levels and subcellularlocalization of IRG1 protein were examined in 78 archived paraffin-embedded glioma samples and 16 normal epitheliumtissues byimmunohistochemical staining. The relationship between the clinic pathologic characteristics and IRG1 expressionin the glioma patients who underwent surgical resection.RESULTSChapter lfirst section:Respectively, as shown in Figure.1-2.the mRNA and protein levels of IRG1 were evidently higher in the U251, SHG-44, TJ-105 and H4 gliomacell lines than the levels in the A251, U118MG and U-373MGcell lines. More importantly, using fresh glioma specimens, we demonstrated that the IRG1 protein level was upregulated in Both si-IRG1-1 and si-IRG1-2 downregulated IRG1 expression, but the inhibitory effect of si-IRG1-2 was more significant in Figure 3a-b and Figure 4.The results of the MTT assays (Figure 5) revealed that the downregulation of IRG1 expression inhibited SHG-44 cell proliferation by 43%(P<0.01), whereas IRG1 overexpression promoted SHG-44 cell growth by 54%(P<0.01). The EDU assay showed (Figure 6.)that overexpression of IRG1 increased the percentage of proliferative cells from ～23 and～25% to ～64 and ～62% in the U251 and SHG-44 glioma celllines; but inhibition of IRG1 using IRG1 siRNA decreasedthe percentage of proliferative cells in the glioma cells (from-23 and ～25% to ～6 and ～9% in the U251 and SHG-44cell lines).si-NC control, the U251 and SHG-44 gliomacell lines transfected with si-IRG1-2 displayed an increasedproportion of cells entering the Gl phase and fewer cells inthe S phase. These results suggestthat the growth-suppressive effect of si-IRG1 was partly dueto G1 phase arrest(Figure 7.). As shown by the colony formation assay,the si-IRGl-transfected U251 and SHG-44 cells formed fewerand smaller sized colonies than did the si-NC-transfected cells((Figure 8.); P<0.01). Cell migration and invasion are integral steps in tumor developmentand metastasis. The wound healing assay showed that thelateral migration of cancer cells was inhibited by IRG1 knockdown (Figure 9.). We found that the knockdown of endogenous IRG1 significantly reducedthe ability of U251 and SHG-44 cells to migrate and invade(P<0.05), compared to the si-NC cells (Figure 10-11). Knockdown of endogenous IRG1 expression induced the activation of the tumor suppressor retinoblastoma protein pRB and the oncogenic cell cycle regulator transcription factor E2F1, while down-regulating the expression of the tumor suppressor cyclindependentkinase inhibitor p21. However, the levels of thecycl independent kinases CDK4 and CDK6 were supressed (Figure 12). In addition, we found that IRG1 inhibition decreasedthe expression of the EMT marker genes snail, N-cadherin, andvimentin, while increasing the level of E-cadherin, anepithelial marker (Figure 13.). The WB result show that knockdownof IRG1 expression by using si-IRG1 decreased the levelsof NF-K B and IkBα, as well as STAT3 (Figure 14.).These results indicate that the abrogation of IRGl expression in glioma cells inhibits NF-K B-mediated signaling.Chapter 1 second section:The average tumor volume in mice injected with the IRG1-depleted U251 cellswas markedly (by>60%) reduced when compared to that inthe control animals (P<0.05) in Figure 15a-b. Tumor weight analysis showedthat sh-IRG1-transfected cells gave rise to significantly smallertumors than did sh-vector-transfected cells (P<0.05) (Figure 16). These results confirmed the previously results in vitro,indicating that expression of IRG1 increases the cancer cellgrowth of gliomas.Chapter 2:Meanwhile, high (defined as greater than median) IRG1 expression inglioma cells was significantly associated with shorter overallsurvival (OS) in the entire cohort (P<0.05) with IRG1 staining.The staining signal of IRG1 was observed mainly in the different stages of glioma tissues (Figure 17C-F) and no signals or only weak signals were detected in the adjacent normal braintissues (Figure 17A-B). The subcellular location of IRG1 wasobserved mainly in the cytoplasm of the cancer cells, occasionallyin nuclei. The result (Table 7) that IRG1 expression was positively correlated with the stageof cancer progression in glioma patients according to TNM stagesⅠ-Ⅱ vs. Ⅲ-Ⅳ (P=0.000). Meanwhile, we stained for IRG1 in glioma tissues in 140 patients. The prognostic effect of IRG1 on glioma patient OS was compared between patients with high and low IRG1 proteinlevels. By Kaplan-Meier curve assessment, patients with a high IRG1 protein level had a significantly lower 5-year survival ratethan those with a low IRG1 protein level. Furthermore, we compared the relationship betweenIRG1 expression and recurrence-free survival (RFS). The patients with IRGl low expression had a long RFS compared with patients with high IRG1 expression. In the patients with TNM stage Ⅱ and Ⅲ, the subgroup with high IRG1 expression had a lower RFS rate than the subgroup with low IRG1 expression (P<0.05).Table 8. IRG1 was highly expressed in 75.6% of the glioma samples compared to only 37.5% of the normal samples, demonstrating a statistically significant difference (P=0.006).CONCLUSION1. The expression of IRG1 was significantly upregulated in glioma compared with normal brain tissue.2.Expression of IRG1 was associated with the TNM stage of glioma patients and important to their prognosis.3. IRG1 is vital to proliferation, invasion and migration of gliomas.4. The mechanisms, including regulation of the cell cycle transition, the EMT pathway and NF-κB signaling suggests that IRG1 may play an important role in the development of glioma.