| Summary: | 博士 === 國立陽明大學 === 醫學生物技術暨檢驗學系暨研究所 === 96 === A comprehensive microarray analysis of hepatocellular carcinoma (HCC) revealed distinct synexpression patterns during intrahepatic metastasis. Recent evidence has demonstrated that synexpression group member genes are likely to be regulated by master control gene(s). My thesis research focused on the functions of and gene regulation by the transcription factor SOX4 in intrahepatic metastatic HCC.
SOX4 plays a pivotal role in tumor metastasis as RNAi knockdown reduces tumor cell migration, invasion, in vivo tumorigenesis and metastasis. A multifaceted approach integrating gene profiling, binding site computation and empirical verification by chromatin immunoprecipitation and gene ablation refined the consensus SOX4 binding motif and identified 32 binding loci in 31 genes with high confidence. The fact that RNAi-knockdown of two SOX4 target genes, neuropilin 1 and semaphorin 3C, drastically reduced cell migration and invasion activity in HCC cell lines suggested that SOX4 exerts some of its action via regulation of these two downstream targets. The discovery of 31 previously unidentified targets expands our knowledge of how SOX4 modulates HCC progression and implies a range of novel SOX4 functions. To characterize the SOX4-affected pathways, microarray gene expression profiling of RNAi knockdown experiments showed that downregulation of SOX4 modulated expression of critical genes involved in regulation of actin cytoskeleton and focal adhesion.
The main theme for gene regulation by SOX proteins depend on requisite partners for target specificity and combinatorial control with partner factors. POU2F1 was identified as a SOX4 co-factor by computational prediction. Whether SOX4 and POU2F1 synergistically regulate NRP1 expression is under intense investigation. RNAi knockdown of SOX4 or NRP1 expression in HCC cell lines, affected expression of epithelial-mesenchymal transition (EMT) related genes. The levels of vimentin, N-cadherin, MMP2 and MMP9 were drastically reduced, but E-cadherin was increased in shSOX4 or shNRP1 treated Mahlavu cells. Our results suggest that SOX4 and NRP1 may play critical roles in liver tumor progression and the signaling pathways mediated by FAK, ERK and Rac are likely to be involved.
Furthermore, we found that liver specific microRNA-122 (miR-122a) was specifically downregulated as human liver tumors developed metastatic potential. Here we presented our initial finding that miR-122a regulates SOX4 expression at the mRNA level. Hence, a new regulatory cascade, from miR-122a through SOX4 to NRP1, was established for liver tumor metastasis. The implication of these results needs to be further examined. This integrated approach sets a paradigm whereby a subset of member genes from a synexpression group can be regulated by one master control gene and this is exemplified by SOX4 and advanced HCC. We provided the relevant gene targets regulated by SOX4 in liver tumors for developing effective diagnostic and alternative therapeutic strategies for intrahepatic HCC.
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