Expression Of Ras In Glioma And Its Effect On The Growth Of Human Glioma Cell

Research background：ras gene was first discovered in the descendants of the Sarcoma virus of Harveryand Kirsten mouse. This gene named ras gene was contained the new gene sequencewhich derived from the host cell’s genome. Since then, people have found the similargene in the bladder cancer (H-ras), lung cance (K-ras) and neuroblastoma (N-ras).Wild type ras gene has the function of tumor inhibition, but it can be actived by genepoint mutation, massive expressions, gene insertion and translocation. Throughtranscription and translation, the actived Ras protein participated in process ofoccurrence and development of tumor. Ras, as an essential signal molecule, isconsidered to be active when bound to GTP and inactive when bound to GDP.Interaction of activated Ras with c-Raf initiates the activation of MAPK(mitogen-activated protein kinase) signaling pathway in a GTP-dependent way. C-Raf is thebound of Ras signaling and MAPK signal pathway, which possessing kinase activity.Activated c-Raf can then selectively phosphorylate MAPK kinase, part of which canundergoes nuclear translocation and phosphorylate transcription factors that controlgenes expression. Besides, abnormal activation of PI3-Kinase/Akt and GEFs/Ralsignals by Ras lead the normal cells lose their capability of apoptosis anddifferentiation, which attribute to excessive cell proliferation and oncogenesissubsequently. In recent years，many research suggested that the actived Ras was veryclose relationship with tumor’s development.In this study, we made use of bioinformatics to gather genes interrelated with Ras in the glioma. And establish the Ras biologic association network, and identifiedseveral interactions of this network with the Ras pathway. Our research not onlyexamined the expression of Ras and Hub genes but also confirmed inhibition ofactived-Ras reduced expression of multiple oncogene in vitro studies. Our studiesprovide strong support for the conclusion that cancellation of actived-Ras specificallyregulates defective Ras pathways in glioma. And last, for Ras as glioma diagnosis andtherapeutic targets to provide the evidence.Methods：1. Bioinformatics methods (NLP analysis, Gene ontology analysis, Pathway andgene network analysis) gather genes interrelated with Ras in the glioma. Andestablished the Ras biologic association network, selected the Hub gene,identified several interactions of this network with the Ras pathway.2. Immunohistochemistry staining tests the positive rate of Ras protein in normalbrain and different grade glioma tissues.3. RT-PCR detects expression of Ras and Hub gene mRNA in normal brain anddifferent grade glioma tissues.4. To inhibit Ras, selecting human glioma cell line U87and U251in thelogarithmic phase and treating with FTS. MTT assay examine U87and U251cellproliferation, then, acquiring the consistency of50%cell survival rate.5. Flow cytometry test the effect on the cell cycle and apoptosis of human gliomacell when the Ras protein was suppressed.6. Western Blot measure the expression of Hub genes when the Ras protein wassuppressed.Results: 1. We utilized text-mining of MEDLINE abstracts with natural language processingto gather genes interrelated with Ras in the glioma. A total of439genes thatinteracted with RAS were identified, and393,96and52genes interacted withKRAS, NRAS and HRAS, respectively. And established the Ras biologicassociation network, selected the Hub gene (PIK3CA, MDM2, CCND1, EGFR,JUN, MYC, VEGFA, ERK1/2), identified several interactions of this networkwith the Ras pathway.2. In different grade glioma, the positive expression rate of Ras protein is73.3%.And in normal brain is only10%. Compared to normal tissue and low gradegliomas(I-II), the mRNA level of Ras and Hub genes expression in highergrade(III, IV) gliomas tissues was remarkably conveyed.3. With FTS treated, the cell survival rate of U87and U251decreased by increasingconcentrations of drug. And with the passage of time in the consistency ofhalf-survival glioma cell livability was in decline.4. Suppression of Ras protein by FTS could inhibited cell growth, arrested the cell atG0/G1phases, and inuced cell apoptosis in U87and U251GBM cell.5. By FTS processing, the expression of Hub genes’ protein, interrelated with Ras,was in decline.Conclusions:1. Gene/protein interaction networks provide critical information for a thoroughunderstanding of cellular processes. Here, we extracted gene/protein interactionsby data mining MEDLINE abstracts and constructed the Ras biologic associationnetwork. Furthermore, PIK3CA、MDM2、CCND1、EGFR、JUN、MYC、VEGFA、ERK1/2were identified as key Hub genes in Ras related genes network. 2. The expression of Ras protein and Hub genes mRNA in glioma was increased andmarkedly correlated with its pathological grade．Downregulation of RAS lead toglioma cells growth suppression, arrested the cell at G0/G1phases, and inucedcell apoptosis in U87and U251GBM cell. And through inhibiting Ras proteinactivity, the expression of Hub genes’ protein was in decline.As a consequence of the above, we considered that inhibiting the Ras activity mayreduce the incidence of gliomas. FTS can be as a potential means of glioma therapy,with important clinical applications.