Down-regulation Of GRIM-19 Promotes The Growth And Migration Of Glioam Cells

Gliomas represent the most frequent primary tumour of intracranial neoplasms and are graded as low-gradeⅠandⅡand high-gradeⅢandⅣaccording to their histopathological and clinical features. Unfortunately, low-grade gliomas will inevitably progress toward malignant high-grade gliomas. Despite the maximal therapies, patients bearing high-grade gliomas still have a median life expectancy of less than 1 year. Accumulating evidence has shown that there are notable analogues between normal development and tumourigenesis. For example, NF1, a tumour suppressor gene, is important for the development of the central nervous system (CNS) and its inactivation has been involved in glioma formation. Sonic hedgehog-Patched signaling pathway plays a critical role in regulating the growth in the normal cerebellum development and is also a major target of mutation in the cerebellar tumour medulloblastoma. Glioma is a classic model that links between tumourigenesis and development. Based on these findings, genes that are involved in normal developmental processes, if dysregulated, might contribute to tumourigenesis. GRIM-19 (Genes associated with Retinoid-IFN-induced Mortality), a novel gene, was initially identified in interferon-β(IFN-β) and all-trans-retinoic acid (RA)-induced tumour cell death pathway using antisense knock-out technique. The GRIM19 mRNA is slightly induced by IFN-βalone. RA alone did not induce the gene expression. However, the combination of IFN-β/RA robustly induced GRIM-19 expression, which leads to apoptosis. It does not belong to any of the known death gene catergories such as of Bcl-2, caspase and death receptor families. Furthermore, we found that homologous deletion of GRIM-19 led to embryonic lethality, suggesting that GRIM-19 is essential for normal embryonic development. GRIM-19 is mainly located in mitochondria with only a small fraction in nucleus. It has multiple functions on cell growth, embryonic development and mitochondria. The GRIM 19 gene maps to a locus on human, chromosome 19p13.2, a region whose loss was associated in cancer progression. Although dysregulated expression of GRIM-19 has been found in some tumours such as renal carcinoma, thyroid tumour and cervical cancers, its underlying mechanisms in tumourigenesis are still poorly investigated. Here, we evaluated the expression of GRIM-19, a novel gene essential for normal development, in various grades of gliomas and several human glioma cell lines. We showed that GRIM-19 mRNA and protein expression were markedly lower in gliomas than that in control brain tissues and negatively correlated with the malignancy of gliomas. Down-regulation of GRIM-19 in glioma cells significantly enhanced cell proliferation and migration, whereas over-expression of GRIM-19 showed opposite effects. We further demonstrated that the activation of STAT3 and the expression of many STAT3-dependent genes were regulated by the expression of GRIM-19. In addition, GRIM-19 exerted its role also probably via non-STAT3 signaling pathway. Collectively, our data suggest that most gliomas expressed GRIM-19 at low levels, which may play a major role in tumourigenesis in the brain. PartⅠExpression of GRIM-19 in human glioma samples.To detect the expression of GRIM-19, we collected 58 clinical speciments from patients diagnosed with primary gliomas. Pathologist certified all these tumor samples used in these studies. These glioma tissues are classified according to the WHO classification standard. There were 16 low-graded (Ⅱ) and 42 high-graded (Ⅲ,Ⅳ) glioma samples. Total 5 normal brain tissues were obtained as normal controls from surgical resections of trauma patients. In this part, total RNA and protein from 58 glioma specimens were extracted using the Trizol reagent and RIPA lysate recommended by the manufacturer. Expression of GRIM-19 was normalized to the levels ofβ-actin mRNA and protein using semi-quantitative RT-PCR and Western Blot analysis, respectively. The RT-PCR result showed that GRIM-19 mRNA levels were lower in malignant glioma than normal brain tissue. In addition, GRIM-19 mRNA levels were inversely correlated with the malignancy of glioma. The Western blot results confirmed the results of RT-PCR. In order to investigate the expression and subcellular localization, the distribution of GRIM-19 was also evaluated in various grades of glioma samples using immunohistochemistry. The results of immunohistochemistry showed that GRIM-19 expression was mainly localized in the cytoplasm of glioma cells. Most cells in normal brain tissue were GRIM-19-positive staining. In contrast, glioma tissues contained less GRIM-19-positive cells than that in normal brain tissues. As grade of gliomas gradually ascends, the GRIM-19-positive cells become gradually decreased. These above results suggested that expression of GRIM-19 is lower in gliomas than normal brain tissues and its expression levels were inversely correlated with grades of glioma. PartⅡThe effect of GRIM-19 on the growth and migration of glioma cells.Although dysregulated expression of GRIM-19 has been found in some tumours such as renal carcinoma, thyroid tumour and cervical cancers, its underlying mechanisms in tumourigenesis are still poorly investigated. To demonstrate the relevance of GRIM-19 to the growth of glioma cells, we have used U251maligant glioma cells (MG), A172 and U87MG three human cell line models. In these studies, we firstly compared the expression of GRIM-19 among these cell lines. The U251MG, A172 and U87MG cells expressed GRIM-19 at relatively high, moderate and weak level, respectively. In the next experiment, GRIM-19 expression was suppressed in U251MG cells with GRIM-19 siRNA and over-expressed in U87MG cells with pXJ40-GRIM-19. The results of Western blot confirmed that GRIM-19 was successfully down-regulated in U251MG cells and up-regulated in U87MG cells at protein levels. Then we observed the growth, colony formation rate and migration of glioma cells under the condition of GRIM-19 up-regulation and down-regulation respectively. The cell viability was measured by MTT assay. Our results showed that the cell viability was promoted in U251MG cells with down-regulation of GRIM-19, whereas the cell viability was suppressed in U87MG cells with up-regulation of GRIM-19. Furthermore, GRIM19 over-expression significantly induced apoptosis of U87MG cells transfected with pXJ40-GRIM-19 compared with control cells. Additionally, colony formation rate of U251MG cells with down-regulation of GRIM-19 were higher than control cells, whereas U87MG cells with up-regulation of GRIM-19 had the opposite effect. These data suggested that down-regulation GRIM-19 promoted the growth and colony formation rate of glioma cells, up-regulation of GRIM-19 had opposite effect. In the injury-induced migration assays, the wound healing capacity was significantly enhanced in U251MG cells with GRIM-19 siRNA compared with control cells. However, the wound healing capacity was drastically suppressed in U87MG cells with pXJ40-GRIM-19 vector compared with control cells. These results suggested that over-expression of GRIM-19 suppressed glioma cell migration induced by injury signal.In conclusion, down-regulation of GRIM-19 promoted growth and migration of human glioma cells. In light of most glioma samples expressing GRIM-19 at low level, down-regulation of GRIM-19 may be involved in glioma carcinogenesis.PartⅢThe molecular mechanism of GRIM-19 involved in the proliferation and migration of glioma cellsTo explore the mechanism of GRIM-19’s roles in the growth and migration of human glioma cells, U251MG and U87MG cells were transfected with GRIM-19 siRNA and pXJ40-GRIM-19 plasmids, respectively. We and other groups have recently demonstrated that GRIM-19 interacts with transcription factor STAT3 and suppresses its ability inducing the genes involved in cell growth promotion. The activation of STAT3 protein is rapid and transient in normal cells, whereas it is persistently activated in numerous human tumors and cancer cell lines. Indeed, in this study, the constitutive activation of (STAT3 tyrosine phosphorylation) PTyr(705)-STAT3 was found in glioma tissues and positively correlated with maligancy grade of gliomas, suggesting that aberrant STAT3 plays an important role in glioma progression. Moreover, the level of PTyr(705)-STAT3 is inversely correlated to GRIM-19 in human glioma samples. Therefore, we examined the effect of GRIM-19 on the expression and activation of STAT3. Western blot results showed that the total protein level of STAT3 remained relatively unchanged in cells regardless of GRIM-19 up-regulation or down-regulation. In contrast, PTyr(705)-STAT3 in GRIM-19 depleted group was signifcantly enhanced compared with the control group, whereas PTyr(705)-STAT3 was evidently decreased in pXJ40-GRIM-19 transfected group. To confirm the results of the Western blot, we examined the effects of exogenous GRIM-19 on the PTyr(705)-STAT3 using double-labelled immunofluorescence in U251MG glioma cells. The results showed that most STAT3 translocated from nucleus to the cytoplasm in pXJ40-GRIM-19 transfected cells compared with the control. In contrast, nucleus translocation of STAT3 from cytoplasm appeared in GRIM-19 depleted cells. Both cytoplasm and nucleus staining of STAT3 remained relatively unchanged in cells transfected with the control vector compared with non-transfected cells. All these results showed that GRIM-19 negatively the phosphorylation of STAT3 and had no effect on the total protein levels of STAT3. Consistently, overexpression of GRIM-19 suppressed a serial of STAT3 target genes including Survivin, cyclinDl, BC1-XL, Bcl-2, Hif1-αand MMP-9, whereas depletion of GRIM-19 enhanced their expression at mRNA level accordingly. All these data indicated that the role of GRIM-19 in glioma is at least partly through STAT3 pathway in glioma cells. In addition to STAT3-dependent pathway, GRIM-19 inhibited glioma cell growth probably via STAT3-independent pathways. To address this issue, we explored the effect of GRIM-19 on two STAT3-independent genes, GADD153 and COX-2. Interestingly, GRIM-19 depletion evidently promoted the expression of GADD153 but suppressed the expression of COX-2. GADD153 has been demonstrated to be associated with apoptosis in response to a number of stress stimuli, including anticancer agents, retinoic acid and nutrient deprivation. COX-2, another STAT3-independent gene, is an inducible enzyme responsible for prostaglandin production and performs important functions in tumorigenesis. Previous reports revealed that COX-2 protein is over-expressed in the majority of gliomas and involved in inhibition of apoptosis. Taken together, GRIM-19 induced glioma cell apoptosis at least partly through STAT3-independent mechanism such as GADD153 and COX-2.In conclusion, we have provided evidence for the first time that GRIM-19 down-regulation occurs in glioma samples and GRIM-19 suppresses glioma cell growth and migration. Additionally, we demonstrated that GRIM-19 in glioma cells is exerted through both STAT3-and STAT3-independent pathways. Our studies may shed light on genes that regulate proliferation, death and differentiation in the nervous system. In addition, considering the role of GRIM 19 in glioma pathogenesis, restoration of GRIM19 may yield a new strategy to treat gliomas.