Nervous System-specific Expression Gene Human Neurotrimin On The Glioma Inhibition

In our previous research, we discovered a novel gene from cDNA library with high identitywith rat Neurotrimin, so we named it Human Neurotrimin (HNT). HNT is highlyexpressed in brain. It belongs to the immunoglobin protein super family. HNT protein is aGPI protein with three Ig domain but transmemberane region and cytoplasmic region. Theresent report shows that HNT has two functions: cell-cell adhesion and synapse formation.We discover its down regulation in dot hybridization and suppose that it may be a tumorsupressor gene.Initially, in 20 glioma tissue samples and 6 glioma cell lines, we confirm with RealtimePCR that HNT is down regulated in 70%(14/20)glioma tissue samples and all of six celllines. We think it is most likely to be caused by epigenetic modification, so we treat gliomacell lines with methylation transferase inhibitor (5-aza-2-deoxycytidine) and deacetylataseinhibitor (TSA), and find HNT expression restores with 5-aza-2-deoxycytidine treatment.As a result, perhaps the CpG islands methylation in HNT promoter region is the cause fordown regulation. Therefore, we demonstrate with MSP and bisulfite sequencing that theCpG island in HNT promoter region is partly methylated in T98G cell. In order to provethat it is methylation lead to down regulation, we seek the core promoter region of HNTwith Luciferase Assay and prove that -606～-136bp is the core promoter region of HNT.We also intend to explore the Sp1 activation to HNT transcription as well as the furtherstudy about mechanism of HNT transcription regulation.Moreover, we investigate the effect of HNT to glioma. We infect U251 cell with emptyadenovirus (Ad) and adenovirus with HNT (Ad-HNT), and discover that U251 cells arearrested in G1 phase after infected with Ad-HNT. We further explore the possible CDK andCDKI that regulates the cell cycle are affected by HNT. Semi-quantitative PCR showsHNT may up regulate p21, p16 and p19, and western blot suggests p21 and p27 are to beup regulated. Moreover, we examine the level of GTP·CDC42 and discover activated CDC42 is down regulated by HNT, so is cyclin E. As a result, it implies that HNT maysuppress glioma via cell proliferation inhibition.