Study On Novel Glioma Development Relative Genes

Exploiting the transcriptional regulation mechnism by microarray and bioinformatics is a important method at genomic research field, and it is better than those methods before. Inhere we analyses the glioma gene expression profiles by clustering and bioinformatic retrieving to find some tumor relative genes that maybe therapy targets in the future, we extracted 28 genes out of 2 gliomas development relative gene expression profiles, validated 7 of them by RT-PCR methods and at last analysis the fuctional changes of Tob gene, one of the potential tumor suppressor genes in Human glioma cell line SHG-44 after transfection.Part I: Bioinformatic Analysis of Glioma Development Relative Genes Objectives: To extract glioma development relative genes by microarray and Bioinformatics that maybe potential gene target used in the future. Methods: Firstly, analysis the glioma gene expression profiles by clustering, and select 2 group of genes that are expressed similarly. Secondly, We search the genomic information about those genes and exploit the relation with the development of glioma. Results: We found 2 groups of gene that is expressed similarly during the gloma development(total 17 genes), one group are upregulated, the others are downregulated, and classified 115 differential expressed genes into 6 classes by Hierarchical Clustering, and extracted 11 genes out of those by Bioinformatics whose functions are related with differential induction. Totally we extracted 28 glioma development relative genes that maybe developed for therapy targets in the future. Conclusions: Bioinformatics is a good tool for analysis of gene expression profile, and we extract some potential key genes by this method.Part II: Analysis of Novel Glioma Relative Genes Expression by RT-PCRObjectives: Exploiting the transcriptional regulation mechnism by microarray andbioinformatics is a important method at genomic research field, but those methods need tobe validated for its high pseudo-positivity and pseudo-negativity. Inhere we analyses 7novel glioma relative genes expression in different WHO grade glioma tissues by RT-PCR,in order to find some tumor development relative genes that maybe cloned for purpose of function research in the future. Methods: Firstly, PCR primers are designed by primer3 software following retrieving those genes sequences in GenBank by their accession number, and then 22 samples of different WHO grade glioma tissues(WHO I,3;WHO II, 8; WHO III, 7; WHO IV 4.) are analyzed by RT-PCR . Finally, the expression level are quantified by SmartView Image processing software. Results: We found the expression profiles of those gene are agree with formal results that we have gotten by Microarray before, like that X54304(myosin, light polypeptide) and AI264216(CTL2 gene) are up-regulated with tumor grade development, NM-019896 (DNA polymerase epsilon p12 subunit), NM₀15962 (CGI-35 protein), AB037886 (NESH protein), D38305 (Tob protein), M87507 (Caspasel) are down-regulated with tumor grade development. Conclusions: The expression profile of those 7 genes gotten by RT-PCR are agree with formal Mocroarray and Bioinformatics results, showing that our Microarray technique are reliable. The expression pattern advised that there are a routine regulation behind the gene networks, and some genes maybe important regulators in the development of gliomas. Part III: Functional Changes of SHG-44 Cells Transfected by Tob Gene Objectives: To study the function of Tob in Human glioma cell line SHG-44 by Lipofectamine transfection. Methods: Firstly We cloned Tob gene by RT-PCR with primers designed according Tob's GenBank record, then constructed its expression vector by linking its CDS fragment with Plasmid pTracer-EF/V5-His A, and at last introduced this recombination DNA into SHG-44 cells by Lipofectamine. The function changes of SHG-44 are analysised by Florence microscope, GFAP Dye and FlowCytometer. Results: Dual enzyme Digest and DNA sequencing showed that the Tob gene have been cloned and constructed into expression vect...