| Glioma is the most common and intractable brain tumor, which has became aserious threat to human health, and malignant gliomas accounted for80%of allprimary malignant brain tumors. Glioma patients usually have poor prognosis, highmortality, high recurrence rate of70%-90%even after total surgical resection. Themedian survival time of glioblastoma and anaplastic glioma were only12-15monthsand2-5years, respectively, and the5-year-survival rate was only4.7%and25.9%,respectively. Despite the application of new technologies and drugs, as well as themuch progress achieved in comprehensive treatment combined of surgical operation,radiotherapy, chemotherapy, immunotherapy and gene therapy, the prognosis ofmalignant glioma patients have not been fundamentally improved. Thus, elucidationof molecular pathological machanism of glioma and discovery of new therapeuticsand molecular targets related with prognosis have become the forefront in this field ofneurosurgery.The malignant transformation of glioma is the result of constantly accumulatedgenetic aberrations and abnormal regulation of growth factor signaling pathways.Several pathways, including the PI3K, p53, RB, MAPK pathways, and many tachessuch as tumor cell adhesion, proliferation, migration, tumor matrix degradation and soon are involved in this process. However, the exact mechanism remains unclear.Therefore, the breakthrough of glioma treatment is to find the key upstream regulatorscorrelated with proliferation and aggressiveness of glioma in these mis-regulatedpathways, and therapies targeted to these key regulators provide a new way of gliomatreatments. Therfore, digging out molecular targets which are helpful to not onlysetting auxiliary radio-chemotherapy strategies, but also predicting prognosis hasgreat clinical significance in order to realize the true sence of molecular targetedtherapy.The arrival of high-throughput technology undoubtedly lays a potent foudationfor the realization of molecular targeted therapy. The usage of high-throughputtechnologies including next-generation sequencing, gene microarray and tissuemicroarray can not only analyze tumor samples, distinguish the subtype of tumors, butalso predict prognosis of disease and the response to drugs and radiation, and thenimplement individual treatments. In the process of treatment, we found that there aregreat differences between the treatment effects of glioma patients with the same pathological type and grade even using the same treatments. The intrinsic molecularvariance of tumors is the most critical factor lead to this phenomenon. If the changesof tumorous gene phenotype such as mutation, deletion could be prefound beforetreatments, and then the therapeutic effects and the prognosis of glioma patients couldbe predicted, it would undoubtedly provide more therapuetic selections and pertinence.It has been reported that the variation of several genes such as the methylation ofMGMT promoter, the mutation of IDH1and the deletion of1p19q could be used topredict the response of some glioma subtypes to chemotherapy drugs such astemozolomide, and could also be used to assess the prognosis.With the in-depth study of cancer at the molecular level, more and moreevidences have demonstrated that post-transcriptional regulation of specific mRNA isclosely related to the expression of several tumor-related genes. Importantly, thisregulation process is conducted via the regulation of cis-acting3’-untranslated regionelement. The cytoplasmic polyadenylation element (CPE) is one of the knownelements which is expressed in hundreds of mRNAs related to cell proliferation anddifferentiation. The CPE binding protein (CPEB) is a group of highly conserved RNAbinding proteins which contain RNArecognition sequences and zinc finger structures.Once binding to the RNAs via CPE, the CPEBs can promote the process of translationinduced by polyadenylation, which promote stem cell development, differentiationand aging processes. The CPEB4is a member of the CPEB family. High expression ofCPEB4has been found in pancreatic cancer and related to higher malignancy andpoorer prognosis. Similarly, the CPEB4is also over-expressed in the GBM. However,little was known about the correlation between CPEB4expression level and thedegree of glioma malignancy, as well as development, proliferation, invasion andprognosis. Thus, in the current study, we will first clarify the expression level ofCPEB4in different pathological grade glioma tissues and its relationship withprognosis of glioma patients via high-throughput tissue microarray, then, we constructlentiviral-mediated RNAi system targeted CPEB4to study the role of CPEB4ontumor proliferation, colony formation, cell cycle and apoptosis in glioma cells in vitro,and explore the possible molecular machanism. Part I. Expression of CPEB4in different grade of gliomas in tissuemicroarray and its relationship with prognosisObjective: CPEB4is an important member of the CPEB family and is involved incell mitosis, meiosis and aging process in both physiological and pathologicalenvironment. Its important role in post-transcriptional regulation indicates thatCPEB4might be closely ralated to tumorigenesis. But few researches have been donein gliomas. In this section, we utilize high-throughput tissue microarrays andimmunohistochemical methods to study the expression level of CPEB4in differentpathological grade glioma tissues and its relationship with prognosis of gliomapatients.Method:1)300cases of pathology-confirmed glioma tissue samples obtained viasurgical resection and16cases of normal brain tissue samples obtained throughtrauma decompression surgery both in the department of Neurosurgery ofChangzheng Hospital, Second Military Medical University from January2000toDecember2010were collected;2) Tissue microarray and immunohistochemicalmethods were uesd to pinpoint the location of CPEB4in both glioma and normalbrain and analyze the relationship between CPEB4protein expression level andpathological grade of gliomas;3),300glioma patients were under long termfollow-up in order to collect the survival data and the relationship between CPEB4expression level and the lifetimes of glioma patients were analyzed.Results: In normal brain tissues, CPEB4was mainly deposited in neurons rather thanglial cells, and its expreesion was at a low level. On the contrary, CPEB4expressionin glioma cells was significantly higher than that in normal brain (P<0.05). In addition,increased expression level was correlated with elevated glioma pathological grade (P<0.05). The results also showed the tight correlation between CPEB4expression andpatients prognosis. The Log-rank univariate analysis showed that longer overallsurvival (OS) and progression-free survival (PFS) were observed in all gliomapatients with lower CPEB4expression level, and vice versa (both P value were<0.001). But after the adjustment by pathological grade, P values were0.065and0.059, respectively. In primary GBM patients, the Log-rank univariate analysisshowed that patients with higher CPEB4expression level had significantly shorter OSand PFS than those with lower CPEB4expression level (both p values were0.011).Cox multivariate analysis showed that CPEB4was an independent prognostic factorsfor OS and PFS in primary GBM patients (OS: HR=2.017, p=0.003; PFS: HR=1.810, p=0.009).Conclusion: In normal brain tissue, CPEB4was expressed in neurons at a quite low level. In glioma cells, CPEB4was mainly expressed in the cytoplasm, and itsexpression level was closely related with glioma pathological grades. These datasuggested that CPEB4may participate in glioma carcinogenesis and invasion. Inprimary GBM patients, CPEB4expression level was significantly correlated withprognosis and was an independent factor to predict the prognosis of GBM patients.Thus, CPEB4is a potential prognostic target for GBM. Part II: In vitro study of CPEB4on the regulation of proliferation ofglioma and its possible molecular mechanismObjective: In the previous section, we have shown that CPEB4was highly expressedin glioma tissues and its expression level is positively correlated with increasedpathological grade of glioma. These data suggested that CPEB4may be related to themalignant biological behavior of glioma. In this section, we investigated the role ofCPEB4on oncogenic behavior of glioma cells in vitro and the possible machanism.Method:1) CPEB4-targeted siRNA sequences were designed and synthesized, thenthey were connected with lentivirus. After that, fluorescence microscopy, real-timePCR, Western blot were used to verify the effects of RNA interference;2) T98gliomacell lines were transfected with CPEB4targeted lentiviral-mediated RNAi andover-expression system. MTT assay was uesd to detect cell proliferation and colonyformation, flow cytometer was uesd to detect cell cycle and apoptosis.3) RT-qPCRwas used to measure the expression level of potential related moleculars.Results: Compared to the control group, the expression levels of endogenous CPEB4were significantly changed after CPEB4-targeted RNA interference andover-expression lentiviral vector were transfected into glioma cell lines. In T98glioma cells, knocking-down of CPEB4resulted in delayed cell proliferation and cellcycle arrested in G1phase. After CPEB4was knock down, SMAD3,PIK3CA andIGF2mRNAwere decreased.Conclusion: CPEB4played an important role in the regulation of the proliferationof glioma cells, SMAD3, PIK3CAand IGF2might be involved in this process. |
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