A Study On The Transformation And Application Of Ependymal Tumors Molecular Classification And Oxymatrine Inhibits Proliferation And Migration And Induces Apoptosis In Human Glioblastoma Cells

?Objective?Molecular diagnosis has been proven of considerable assistance in tumor clinical diagnosis,treatment and prognosis.Here we presented a study of c11orf95–RELA and YAP1-MAMLD1 gene fusions in supratentorial ependymomas,and infratentorial ependymal tumor PFA and PFB gene expression features in single center tumor samples,and its potential clinical application.In the other part,the antitumor activity of oxymatrine in glioblastoma was preliminarily discussed.?Methods?NanoString nCounter,RT-PCR and qPCR assays were used to detect the expression of fusion genes(c11orf95-RELA and YAP1-MAMLD1),wild-type genes(RELA,L1CAM,YAP1 et al.)and the signature genes of PFA and PFB ependymal tumors.Gene expression results were also compared to magnetic resonance imaging(MRI)features in several posterior fossa ependymomas.In glioblastoma(GBM),effects of oxymatrine(OMT)were revealed by MTT,Transwell,high content screening(HCS),Western blot,and Annexin V FITC/PI double staining in cell lines U251 and A172.?Result?69%(9/13)of fresh frozen tomor tissue samples were determined positive in RELA fusion type 1 and 15%(2/13)in fusion type 2 by NanoString nCounter Analysis.RELA^FUS1 is positive in ST-6D T and ST-6R T cases,negetive in the supratentorial primary tumor sample ST-9D T but positive in the recurrent tumor sample ST-9R T.No fusions were found in DNA.The expression of wild type RELA and L1CAM was up-regulated.The infratentorial samples can be divided into three subgroups by subgroup signature genes:LAMA2(PFA),NELL2(PFB)and LRRC7(PFB),and we also found the expression level of RELA,MAMLD1 were similar to LAMA2.The MTT assay indicated that OMT inhibited the proliferation of glioma cells significantly.Flow cytometry results suggested that OMT(10^-5 M)treatment may induce U251 and A172 cells apoptosis.In addition,western blot test demonstrated significant increase in the expression of bax and caspase3 while decrease of bcl-2 in GBM cells.OMT also can diminish the migratory ability of the indicated cells evaluated by transwell assay and HCS assay.?Conclusion?C11orf95-RELA gene fusion type 1 was detected in 69%of supratentorial ependymal tumor,and type 2 was 15%.The fusions were caused by false splicing of transcripts rather than chromosome rearrangement.And the primary and recurrent tumor can show distinct results,indicating the potential role of gene fusion in tumor recurrence.C11orf95-RELA fusion up regulated RELA and its downstream gene L1CAM,which is a key molecule in NF-?B pathway involved in the carcinogenic process.The expression of signature genes of PFA subgroup(LAMA2,and new finding of RELA,and MAMLD1),and PFB subgroup(LRRC7 and NELL2),combined with imaging(MRI)features,revealed a beneficial method in grouping infratentorial ependymal tumor.We also found that OMT can inhibit proliferation and migration of GBM cells while inducing GBM cells apoptosis.