The Role Of Notch Signaling Pathway In The Mechanism Of Glioma Pathogenesis

Glioma is the most common malignant tumor in central nervous system with thehighest mortality, due to its high degree of atypia, strong invasiveness and chemoradiationresistance, the mortality of glioma is the highest in the cerebral diseases after stroke. Asthe pathogenesis of gliomas has not yet been fully elucidated, there is not effective methodof treatment for gliomas, that’s why the median survival of of patients with the mostmalignant glioma is only14.6months and5-year survival rate is only9.8%, so gliomatreatment has always been the medical challenges faced by the neurosurgeon. Thus, theresearch on glioma pathogenesis is expected to provide a new means of adjuvant therapyfor the treatment of gliomas. In recent years, many studies confirm that there exists a small group of special cellsin glioma, these cells, possessing the characteristics of stem cells, is named glioma stemcells (GSCSs). GSCs have the following characteristics: fast proliferation, with thepotential of multi-directional differentiation, resistant to the chemoradiotherapy, isconsidered to be the main reason for poor effectiveness of glioma treatment and rapidrelapse. Therefore, in addition to the current conventional means of combination therapyfor glioma, looking for the target eliminating the GSCS stem cells can provide a newmeans of adjuvant therapy for glioma treatment by finding core regulatory molecules andkey signal pathways modulating CSCs.Notch signaling pathway is highly conserved during evolution and plays an importantrole in cell proliferation, differentiation, and in the cell fate determination. Studies haveshown that the Notch signaling pathway is activated in gliomas, and may participate in themaintenance of normal neural stem cells and glioma stem cells, regulation of neural stemcells and glioma stem cell self-renewal, promoting their proliferation, inhibit theirdifferentiation. Inhibition of the Notch signaling pathway and deplete glioma stem cells ingliomas; Activation of the Notch signaling pathway and enforce the tolerance of gliomastem cells to radiotherapy and chemotherapy. Therefore, the Notch signaling pathwayplays a vital role in maintenance of the phenotype of GSCs, but the research is still notdeep enough on the exact mechanisms of GSCs regulation by Notch signaling pathway.The preliminary studies in our lab have shown that, KyoT2in mice (which is calledFHL1C in human) can interact with RBP-J, the critical transcription factor of Notchsignaling pathway, to inhibit the Notch signaling pathway, based on the important role ofthe Notch signaling pathway in glioma, we assume that FHL1C could inhibit proliferationof glioma and glioma stem cells and promote the differentiation of glioma stem cells byinhibiting Notch signaling pathway in order to cure glioma. But our western blotexperiments did not detect FHL1C expression in gliomas and normal brain tissuesurrounding glioma, which is inconsistent with our predictable results that FHL1C isnegatively correlated with Notch signaling pathway; However, we found the expression ofFHL1A which is a different subtypes of FHL1C, the expression of FHL1A was significantly higher than in normal brain tissue than in the tumor tissue, this unexpecteddiscovery attract our concern.Through a literature review and our preliminary experiments, we speculated FHL1Amay be a tumor suppressor gene in glioma, we conducted the following experiment：Part1Objective：To explore the expression of FHL1A in glioma specimens and the correspondingnormal brain tissue, and the relationship between FHL1A expression and tumor grade ofglioma, clinical characters and the prognosis of glioma patients; To study the inhibition ofFHL1A on glioma proliferation and reveal the molecules mechanism by which FHL1Ainhibit glioma proliferation; This study will provide a theoretical basis for establishingFHL1A as a potential target of glioma molecular targeted therapy.Methods:1. To study the expression of FHL1A in46patients with gliomas and the normal braintissue surrounding the glioma, by immunohistochemistry and western blot; To detect theexpression of FHL1A in114different grade glioma samples by immunohistochemistry; Tostudy the relationship between expression of FHL1A and clinicopathological features andprognosis of glioma patients.2. To establish U87stable cell line transfected with FHL1A expression Lentiviral vectorand U251stable cell line trasnfected with FHL1A siRNA Lentiviral vector and to study itsimpact on the proliferation of glioma cell lines by MTT assay and flow cytometry.3. To further reveal the mechnism that FHL1A affect glioma proliferation bymodulating PI3K/AKT pathway. In addition, inhibitor of PI3K/AKT pathway, LY294002,can inhibit proliferative effect of FHL1siRNA, this further clarify the mechanism ofFHL1A affect glioma proliferation.Result1. FHL1C expression does not exist in gliomas and the normal brain tissue surrounding, which is contradict with our hypothesis, while FHL1A expression in gliomas is lower thanthe corresponding normal brain tissue around the tumor(p=0.0419＜0.05), FHL1Aexpression is inversely correlated with glioma grade, and low FHL1A expression predictspoor prognosis of the glioma patients;2. FHL1A is highly expressed in anaplastic glioma cell line U251, but no expression isdetected in GBM cell lines U87, Overexpression of FHL1in U87cell line and inhibitglioma proliferation, while downregulation of FHL1by siRNA in U251can promote itsproliferation;3. FHL1A inhibits glioma proliferation by inhibiting PI3K/AKT pathway.Conclusion1. FHL1C may not be involved in the occurrence and development of glioma;2. FHL1A can inhibit proliferation of glioma by regulating PI3K/AKT signaling;3. The mechanisms than Notch signaling pathway regulate GSCs remains to be furtherstudied;4. We speculate that Notch may regulate biological function of glioma through miRNAs?Part II:Our research team performed miRNA microarray in two groups, the first group isneural stem cells(NSCs) spheres from RBP-J knockout mice, and the second group isnormal neural stem spheres from wild type littermates together with RBP-J knockout.RBP-J is key transcription factor to inhibit Notch signaling pathway, so the differencebetween the two groups is whether Notch signaling pathway is inhibited. In all,3pairs and6samples were analyzed. The result indicated that miRNAs differentially expressedbetween the two groups, one of the most obvious difference in the miNAs expression ismiR-342-5p.The miRNA microarray results suggest that inhibition of the Notch signaling pathwaycan up-regulate the expression of miR-342-5p; A miRNA microarray study has shown thatthere are similar miRNAs expression profiling between glioma and NSCs, and some miRNAs regulating NSCs can also participate in the regulation of GSCs.Therefore, we assume that miR-342-5p is also up-regulated by inhibiting Notchsignaling pathway in GSCs, miR-342-5p may be involved in the regulation of malignantglioma stem cell phenotype, that’s why we carried out the following experiment to verifyour assumptions:ObjectivesTo explore the regulation of miR-342-5p expression by Notch signaling pathway in GSCs,different miR-342-5p expression between glioma and its surrounding normal brain tissue,and in glioma of diffetent grade; To reveal the influence of miR-342-5p on GSCsproliferation and differentiation, and then reveal the molecular mechanism thatmiR-342-5p regulate the biological function of GSCs. For the establishment of the Notchsignaling pathway as a target of miR-342-5p, targeting eliminate GSCs and thus achievethe purpose of treatment of gliomas. This study will provide a theoretical basis forestablishing the Notch signaling pathway and miR-342-5p as a potential target of gliomamolecular targeted therapy.Methods1. To analyze different expression of miR-342-5p in glioma and its the correspondingnormal brain tissue and its diffetent expression in different grade gliomas by real-timePCR;2. To establish the stable culture system in vitro for GSCs from fresh specimen ofglioma patients;3. To analyze the expression of miR-342-5p in GSCs and non-GSCs by real-time PCR.To verify the regulation of miR-342-5p by Notch signaling pathway by real-time PCR;4. To study whether and how miR-342-5p can influence proliferation and differentiationof GSCs by immunofluorescence and real-time PCR.5. To verify the regulation of downstream target genes Pokemon (ZBTB7A)bymiR-342-5p by report assay and western blot.Result 1. The expression of miR-342-5p in gliomas was significantly lower than thecorresponding peritumoral normal brain tissue, and the difference has statisticalsignificance (P=0.0157<0.05); The expression of miR-342-5p was inversely correlatedwith tumor grade of gliomas(P=0.0078<0.05);2. Stable culture system of primary cultured GSCs in vitro has been established;3. Inhibiting Notch signaling pathway byγ-secretase enzyme inhibitors (GSI) canup-regulate the expression of miR-342-5p in CSCs compared to not blocking group;4. MiR-342-5p inhibit the proliferation of GSCs and inhibit the differentiation ofGSCs;5. Result from reporter assay and western blot showed that miR-342-5p candownregulate Pokemon (ZBTB7A) by targeting its3’UTR.Conclusion1. miR-342-5p expression in glioma is lower than corresponding normal brain tissuemiR-342-5p was negatively correlated with tumor grade of gliomas;2. miR-342-5p can inhibit the proliferation of GSCs and inhibited differentiation ofGSCs to its offspring neurons and astrocytes;3. miR-342-5p can inhibit proliferation and differentiation of GSCs by inhibiting theexpression of the cancer gene ZBTB7A.4. Notch signaling can modulate malignant phenotype of GSCs through miR-342-5p.