Research Of SOX4’s Function And Its Regulation Network In Glioblastoma

The sex-determining region Y (SRY) box, Sox gene family characterized by the highly conserved HMG-domain responsible for binding sequence specific DNA is comprised of genes encoding transcription factors which are essential for embryonic development and cell fate determination, differentiation, and proliferation. So far, twenty pairs of Sox genes have been identified in human and mouse genomes. SOX4is the member of SOXC group which also include SOX11and SOX12.SOX4has been proved to be involved in the regulation of the development of B cell, lymphocyte, osteoblasts cell and neuron cell. In the past decades, SOX4has been found over-expressed in adenoid cystic carcinoma (ACC), hepatocellular carcinoma, bladder tumors, acute myeloblastic leukemia, prostate cancer, endometrial cancer and glioblastoma.SOX4together with Sox11are critical in the formation of neurons in central neural systems (CNS). In glioma stem cell (GSCs), SOX4is induced by TGF-beta and then trans-activate SOX2which is important for the maintenance of stem cells. Lin. et al has reported that SOX4’s expression in glioblastoma is higher than normal brain tissues on protein and mRNA level, which has been proved by Real-time PCR and IHC. But its role in glioblastoma and the molecular mechanism is not clear. In this work I try to uncover SOX4’s function and its regulation network in glioblastoma cells, which contains four parts:Firstly, I found SOX4expression in Glioma Stem Cells (GSCs) is higher in glioblastoma cells. Functional analyais showed that GSCs appear to grow fast and weaker cell-cell attachment with reduced stem cell marker expression while SOX4been reduced by RNAi.Next, we explored SOX4’s function in glioblastoma cells. We choosed cell lines which have relative lower SOX4expression to other glioblastoma cells, and did transient overexpression or stable SOX4overexpression by lentivirus. Functional analysis showed that SOX4represses glioblastoma cell proliferation and induce G0/G1cell cycle arrest. Knockdown SOX4by Cre-recombinase recorved cell growth ability and reduced G0/G1cell cycle arrest. I further proved this change is caused by SOX4induced p53nuclear accumulation followed by activated p21signaling.In the third part, I used microarray analysis and found633genes regulated by SOX4(fold change>1.8), these genes involved in cell cycle, growth, chromosome, DNA replication, packaging, steroid metabolic. Though I found SOX4up-regulates beta-catenin on mRNA and protein level, it can not promote cell proliferation because beta-catenin failed to translocate into nuclear and can not interact with TCF/LEF in order to induce the down-stream of Wnt signaling.In the last part, I used Chromatin Immunoprecipitation-sequencing to identified SOX4binding target on human genome,18genes from ChIP-seq were also found to be regulated by SOX4in microarray data (fold change>1.5),9of them with fold change>1.8. By comparing with SOX4’s target genes in LNCaP cells, I found44overlapped genes, in which I proved HDAC9as a direct SOX4target by western blot and Real-time PCR.In conclusion, this research proved that SOX4overexpressed in GSCs, knockdown of SOX4in GSCs promoted cell growth and reduced cell-cell attachment and reduced stem cell marker (CD44) expression. In glioblastoma cells, SOX4acts as a tumor suppressor gene which inhibit cell growth by induce G0/G1cell cycle arrest. We proved this suppressor function is caused by induction of p53-p21signaling and is independent with up-regulation of beta-catenin. By integrative analysis with gene expression profiling and ChIP-seq of SOX4in LN229cells, I provide more evidence about SOX4’s role and molecular mechanism in glioblastoma, which are very help for the future work.