AJAP1Abnormal Expression In Glioma And Inhibition Of Cell Proliferation And Invasion Of Molecular Mechanism Research

Glioma is the most common primary intracranial tumor, two-thirds more than formalignant glioma, account for about2%of adult body tumors, highly malignantglioma tumor median surial only1year,5years of survival is almost zero.Surgicalresection, postoperative chemotherapy and radiotherapy curative effect is not veryideal, glioma still is one of the worst prognosis of tumor in systemic tumors, there isno radical cure.The characteristics of glioma invasive growth tends to be the rootcause of glioma is difficult to cure and recurrence.Therefore glioma treatmentstrategy of optimization not only should fix attention on in the treatment of theprimary tumor, still should focus on control to normal brain tissue invasion andmetastasis of tumor cells.Identifying an inhibition of glioma cells characteristic ofinvasion and metastasis of tumor associated genes, further reveals the mechanism,treatment of glioma is of great significance.A growing body of evidence suggests that many similarities in1p36regioncoding tumor suppressor genes.Recent study found that adhesive connectionAssociated Protein1(Adherens Junctional Associated Protein1, AJAP1, also knownas a Shrew-1) as another similar tumor-suppressor genes coding region onchromosome1p36.Radlwimmer team in patients with glioblastoma genomichybridization and microarray analysis comparison, AJAP1first described as a kind ofbiomarkers and glial cell.Subsequently, Zhang and his colleagues confirmed in mostof the cell lines, there are highly methylated AJAP1promoter region and in gliomacells express AJAP1can inhibit cell invasion and metastasis.But, change AJAP1expression level in glioma cells for cell invasion and proliferation of the underlyingmechanisms are still not clear.This topic focuses on AJAP1abnormal expression in glioma, explore therelationship between its and glioma level, application AJAP1restructuring slow virusvector infection technology change the expression of glioma cells AJAP1level,preliminarily study AJAP1influence on glioma malignant biological phenotype andits potential mechanism. MethodsThe first part of the application of immunohistochemical staining andimmunofluorescence confocal imaging methods,5cases of normal brain tissuespecimens were48cases of gliomas in AJAP1expression level, using Western blotmethod for eight glioma cell line,1AJAP1protein expression levels were detected innormal cell lines, preliminary study AJAP1and the relationship between the level ofglioma.The second part focuses on the research and application AJAP1recombinantadenovirus plasmid of human brain glioma cells malignant biological phenotype andin vitro study of the impact of cytoskeleton, and discussed its potentialmechanism.Using AJAP1recombinant adenovirus plasmid infect U87、U251cells,Transewell change detection glioma cells invasion ability in the experiment;Tablet ofclone formation test cell proliferation ability;Scratch the experimental evaluation cellmigration ability.Western blot method to detect the change of glioma cell proliferation,invasion and other related indicators, at the same time after immunofluorescencemethod is applied to detect the expression AJAP1changes of cytoskeleton.The third part is using brain stereotactic technology will also transfection AJAP1recombinant adenovirus and luciferase slow virus U87glioma cells inoculatedBALB/c-the size of a5weeks nu caudate nucleus (SPF) female nude mice toestablish intracranial glioma model, set up blank control group, with livebioluminescent Imaging System (IVIS Lumina Imaging System) dynamicobservation of tumor growth and changes of nude mice survival and histopathologicalmethod is applied to detection of two groups of animal specimens in proliferation,tumor, etc. The change of protein expression levels.ResultsBy immunohistochemical detection of paraffin section showed that theexpression of glioma samples AJAP1level was significantly lower than normal braintissue, and with glioma level increased, AJAP1expression with reduced.Organizationimmunofluorescence test is also confirmed in high-level glioma specimens, theexpression of AJAP1significantly lower than the low grade gliomas specimens, consistent with immunohistochemical results.Therefore AJAP1expression level andnegatively correlated with pathological grading gliomas.In glioma cell line, Westernblot detection in human embryonic kidney epithelial is highly expressed in HEK293cells, and significantly lower expression in the majority of glioma cell line, A172,TJ905, TJ899low expression in the most significant.Then, using the AJAP1recombinant adenovirus infection U87, U251glioma cells, AJAP1protein expressionlevel, by improving the cells AJAP1carrier group and blank control group andtransfection empty vector group compared with flat tumor cell clone formation testshows AJAP1group on the number of clones formed to reduce, transwell experimentsshow AJAP1group cell invasion ability was significantly inhibited, scratch test showthat cells’ ability to migrate to fall, the Western Blot detection of Ki-67, MMP-9protein expression levels were significantly lower.In cell immunofluorescence test,after expressing AJAP1, U87, U251cell skeleton structure change, U87-AJAP1group compared with U87-vector group, cells from the original spherical growth forminto flake, pseudopodia, cells show kind of circular shape.In U251cells, can also beobserved pseudopod disappeared.In vivo animal experiment, live animals of bioluminescence imaging resultsconfirm U87-AJAP1tumor significantly less than U87-vector group, and the vectorgroup all died within three weeks, after three weeks,AJAP1group only deaththree.Immunohistochemical results show that the express AJAP1raised obviously,and the expression of Ki-67、MMP-9significantly lowered, consistent with cellexperiment results.Conclusions1.With the benchmark in normal brain tissue, AJAP1genes in glioma specimensand absence expression in malignant glioma cell line or low expression, andnegatively correlated with tumor grade, suggest possible AJAP1as a tumorsuppressor genes involved in the occurrence and development of the gliomas.2. Application AJAP1recombinant adenovirus vector and infect the human brainglioma U87、U251cell, which can effectively inhibit tumor cell proliferation,invasion and migration ability, cell immunofluorescence after prompt express AJAP1,may through to the reconstruction of the cytoskeleton, pseudopodia disappeared or reduced, thus reducing glioma cell invasion and migration ability.3. Intracranial glioma model, successfully established U87cells in the bodyfurther confirmed AJAP1tumor suppression effect, is consistent with experimentalresults in vitro.4. Through to the AJAP1for glioma proliferation, invasion and migration thepreliminary research on the molecular mechanism of inhibitory effect, show AJAP1can be used as potential targets of glioma gene therapy.AJAP1closely associated withthe cytoskeleton, worth further discussing.