Fragile X Mental Retardation Protein Promotes Astrocytoma Proliferation Via MEK/ERK Signal-pathway

Background Gliomas are the most common type of primary brain tumor, accounting for approximately 80% of all malignant intracranial tumors. Gliomas are subcategorized into four grades(I-IV) on the basis of histopathologic evaluation and clinical criteria. High-grade gliomas, which proliferate aggressively, are associated with poor prognosis. Therefore, obtaining a better understanding of the molecμlar mechanisms underlying glioma growth will be important in the development of novel management strategies to improve patient survival. The proliferation of glioma is strongly associated with the expression of certain functional proteins, such as decoy receptor 3(Dc R3), a member of the tumor necrosis factor receptor super-family, and leucine-rich repeat containing G protein-coupled receptor 5(LGR5). Furthermore, constitutive activation of mitogen-activated protein kinase/ERK kinase(MEK)/extracellμlar signal-regμlated kinase(ERK) signaling has been reported to promote the proliferation and migration of glioma cells. Thus, there is considerable interest in identifying additional proteins that influence the proliferation of glioma, as these may prove to be novel therapeutic targets for the development of improved treatments.As a single gene disease with X-linked condition, the fragile X syndrome(FXS) is caused by FMR1 gene absent or mutated to induce the absence of FMRP. FMRP is an RNA-binding protein that plays an important role in the development of connections(synapses) between nerve cells. The synapses mediate cell-to-cell communication, and these can change and adapt over time in response to experience; this characteristic is known assynaptic plasticity, and is important for learning and memory. FMRP helps to regμlate synaptic plasticity by carrying m RNA molecμles from the nucleus to other regions of the cell where proteins are assembled, and regμlating the translation of these m RNA molecμles to proteins, some of which are important for the normal functioning of neurons. FMRP binds to m RNAs and regμlates translation as well as transport and stability of its targets. It is therefore important to understand which m RNAs are controlled by FMRP. Several approaches are used to identify a lot of FMRP putative target m RNAs(>1000 in brain and >6000 in nonneuronal cells). Many of the FMRP target m RNAs encode for proteins mostly involved in synaptic activity, cell adhesion, and cytoskeleton structure and remodeling. Lack of FMRP leads to the disorder of translation of m RNAs combined with FMRP, and functional protein expression out of control. Patients with FXS show neurological symtoms, including intellectual disability, hyperactivity, obsessive-compμlsive disorder, anxiety, and autistic-like behavior. Although the detailed mechanisms underlying the phenotype in FXS remain to be characterized, it has been reported that the absence of FMPR may lead to dysregμlation of MEK/ERK and phosphoinositide 3-kinase(PI3K)/mammalian target of rapamycin(m TOR)/glycogen synthase kinase-3-beta(GSK-3β) signaling.Research into clarifying the function of FMRP in the brain has revealed a relationship between FMRP expression and cancer. For example, the increased expression of FMRP in breast cancer and hepatocellμlar carcinoma is associated with a more aggressive metastatic phenotype. Interestingly, however, there is evidence that patients with FXS may have an unusually low risk of brain tumor progression. A case study reported that a boy with FXS who developed an inoperable midbrain glioblastoma was still alive and well 8 years after the diagnosis, with no specific neurologic abnormality. It has not been report whether the slow progression of neuroastrocytoma is correlated to deletion of FMRP in FXS. In this study, we focus on the relationship between FMRP and neuroastrocytoma proliferation. We will clarify the molecμlar mechanism of FMRP to play a role in neuroastrocytoma.Materials and Methods1. Patients and tissue samplesWe retrospectively reviewed the electronic medical records and radiology information systems of the No. 2 Affiliated Hospital, Guangzhou University, China to identify patients diagnosed with an astrocytoma between January 2004 and October 2014. A total of 74 patients were included in the study, and serial sections of their astrocytoma were obtained. Among the 74 enrolled patients, 42 had WHO grade II astrocytomas,and 32 had WHO grade III or IV astrocytomas. Grade III astrocytomas and grade IV astrocytomas were classified as high-grade gliomas, while grade II astrocytomas were categorized as low-grade gliomas. The pathologic diagnosis and glial fibrillary acidic protein(GFAP), Ki67 and FMRP status were verified by two different pathologists. Glioma tissue samples were obtained from 24 patients, for use in an additional series of experiments. These patients had undergone surgical resection of their glioma in our institution between March 2013 and March 2014. Additionalbrain tissue samples, which were used as controls, were obtained from 6 patients with cerebral trauma.2. The expression of FMRP in astrocytoma tissues of varying pathologic grades, and analyses of overall survival in patients with astrocytomaaccording to the Ki67 or FMRP status.FMRP was detected by Western blot in brain tissue samples from patients with astrocytoma of varying pathologic grades(II, III or IV) or cerebral trauma(?normal‘). The brain tissue sections from patients with astrocytoma(grades II, III or IV) was stained with H&E or immunostained for GFAP, Ki67 or FMRP. The staining of cells with FMRP was quantified as the integrated optical density(IOD) per field of view, using Image-Pro Plus 6.0 software. Percentage of Ki67+/FMRP+ cells was measured from immunostained brain tissue sections from patients with astrocytoma. Meanwhile, we analysised the relationship between FMRP and Ki67 in astrocytoma. Kaplan-Meier survival curve for patients with astrocytoma, grouped according to the percentage of Ki67+ tumor cells and the IOD of FMRP+ cells.3. The expression of FMRP and proliferation of four astrocytoma cell linesThe detection of FMRP by Westernblot was performed in U251 and U87 cell lines. Proliferation ratios for U251/U87/BT325/SHG44 cells were determined in the experiments.4. FMRP promotes proliferation of astrocytoma cell linesThe U251/U87 astrocytoma cells was transfected by liposome encapsμlated si RNA to target the 3?-UTR of FMR1 gene to silence the expression of FMRP. MTT cell proliferation assay and percentage of Edu-incorporating was performed in U251 or U87 cells, to detecte proliferation ability of astrocytoma cells.5. FMRP induces MEK/ERK activation in astrocytoma cells. Under the condition of FMRP-si RNAs transfected into U251 or U87 cells, western blot was performed to detected FMRP, PTEN, PDK1, AKT, m TOR, GSK-3β, MEK and ERK, and immunofluorescence staining experiments was performed to detected FMRP or MEK in the same cell lines, to illuminate that FMRP induced activation of MEK/ERK signaling pathway in astrocytoma cell lines.6. FMRP promotes growth of astrocytoma xenografts in vivoWestern blots showed FMRP expression in U251 cells that were either infected or uninfected with lentivirus vector carrying GFP-sh RNA or FMRP-sh RNA. U251 cells were inocμlated into the pads of nude mice to develop orthotopic tumors. Relative tumor volumes(RTV)were measured at the indicated times. The sections of orthotopic tumors from mice, stained with H&E or immunostained for GFAP, Ki67 or FMRP. The xenograft tumors were derived from U251 cells either infected or uninfected with lentivirus vector carrying GFP-sh RNA or FMRP-sh RNA.7. Statistical analysisAll statistical analyses were performed using SPSS for Windows version 19.0(SPSS Inc., IL, USA).Result1.FMRP expression in astrocytoma correlates with tumor proliferation and prognosis of patientsWestern blot was used to detect FMRP protein expression in tissues amples from 24 patients with astrocytoma of varying grades and 6 patients with cerebral trauma(as a control). Compared with the control, the protein level of FMRP was increased about 5.1-fold in grade IV astrocytoma(P<0.05) and about 3.2-fold in grade III tumor(P<0.01). IHC(in tissues from a different group of 74 patients) revealed that as the astrocytoma grade increased, GFAP expression decreased, whereas the levels of FMRP and Ki67 markedly increased. The proportion of Ki67+ cells or FMRP+ cells were increased in grade III and grade IV disease(P <0.05 or P<0.01) compared with grade II. The percentage of FMRP+ cells in Ki67+ rate ≤5%, was increased compared with Ki67+ rate > 5%(P<0.01). These data imply that astrocytoma cell proliferation correlates with FMRP expression. Kaplan-Meier survival analysis revealed significantly improved survival in patients with a low percentage of Ki67+tumor cells(P < 0.001) or low IOD of FMRP+cells(P< 0.01). These data indicate that high FMRP expression is associatedwith faster growth of the astrocytoma,and the poor prognosis of astrocytoma patients.2. FMRP may promote proliferation of astrocytoma cells via MEK/ERK signalingThe relationship between FMRP expression and tumor proliferation was further investigated in astrocytoma cell lines. The resμlts revealed that FMRP higher expression was correlated with proliferation of astrocytoma cell lines(P <0.01). Transfection of cells with two different FMRP-si RNAs(separately) resμlted in decreases in proliferation(measured with the MTT assay) of 33.3% and 43.0% in U251 cells(P < 0.05 and P < 0.01 vs. untransfected cells), and 37.1%and 44.1% in U87 cells(P < 0.05 and P < 0.01 vs. untransfected cells), respectively. Similar resμlts were obtained using Edu incorporation to assess cell proliferation. Collectively, these data suggest that FMRP may play an important role in the proliferation of astrocytoma cells.Further experiments were performed to explore the effects of decreased, rather than absent, FMRP expression on signaling pathways. Transfection of either of the two FMRP-si RNAs into U251 or U87 cells did not alter the phosphorylation of PTEN at Ser308, PDK1 at Ser241, AKT at Thr308 or Ser473, GSK-3β at Ser9, suggesting no effect on AKT/m TOR/GSK-3β signaling. Interestingly, both FMRP-si RNAs inhibited total MEK levels and reduced the phosphorylation of MEK at Ser217/221 and ERK at Thr202/Tyr204; GFP-si RNA and mock transfection were without effect. Immunostaining experiments also revealed that MEK expression in U251 cells was reduced by both FMRP-si RNAs, with no effect of GFP-si RNA or mock transfection. Taken together, these data imply that FMRP may influence astrocytoma cell proliferation via the MEK/ERK signaling pathway.3. FMRP promotes proliferation of astrocytoma in vivoU251cells were either infected or uninfected with lentivirus vector carrying GFP-sh RNA or FMRP-sh RNA, and inocμlated into the pads of nude mice to develop orthotopic tumors. The volumes of U251 cell xenografts infected with lenti-FMRP-sh RNAwere reduced by 37.2% at day 9(P < 0.05),44.2% at day 12(P < 0.05), and 36.3% at day 15(P < 0.01), as compared with uninfected xenografts. Furthermore, the weight of the tumor derived from cells infected with lenti-FMRP-sh RNAwas reduced by 61.5% on day 15(P < 0.01).Immunohistochemistry experiments on orthotopic mouse tumors revealed that xenografts from U251 cells infected with lenti-FMRP-sh RNA exhibited decreased expressions of FMRP and Ki67, and increased expression of GFAP. Overall, lenti-FMRP-sh RNA was associated with a 34.7% decrease in Ki67expression(P < 0.05) and a 77.1% reduction in FMRP expression(P < 0.05). All these data indicate that FMRP promotes proliferation of astrocytoma in vivo.ConclusionIn summary, our study shows that increased expression of FMRP, an important RNA binding protein, promotes astrocytoma proliferation via MEK/ERK signaling pathway.1.The expression of FMRP and Ki67 increases along with the elevation of histopathologic grade in astrocytoma. The level of FMRP increases in the higher pathologic grade of astrocytoma. FMRP is correlated to the proliferation of astrocytoma.2.The expression of FMRP correlates with the prognosis of astrocytoma patients. The elevated expression of FMRP in glioma induces poor prognosis of astrocytoma patients.3.FMRP promotes the proliferation of astrocytoma cells. Enhanced FMRP expression in astrocytoma may increase the expression of MEK, to promote proliferation through activation of MEK/ERK signaling.4.Furthermore, the expression of FMRP in astrocytoma xenografts of nude mice correlates to the expression of Ki67. FMRP promotes the growth of astrocytoma xenografts.