PAF复合物在肝癌细胞中的功能及其机制研究

Aberrant expression of the subunits in RNA polymerase II associated factor complex (PAFc) is ubiquitous during development and progression of human cancers.PAFc is involved in human leukemogenesis through interaction with a rearranged protein MLL-AF9. Our works determined PAF1c acts as an arginine methyl histone effector that mediates transcriptional activation of estrogen receptor target genes. While, the functions and mechanisms of PAF1c in hepatome cells are still elusive. Exploring subset of PAFc target genes that program the cancer cell phenotype will exert deep impacts on our understanding of of PAFc's function in tumor cells, even on development of new therapy targets. In this project, a cell line, that the expression of endogenous Paf1 is replaced by Flag-tagged Paf1, will be constructed for identification of the interactome of PAFc by co-IP methods using anti-Flag antibody. ChIP-seq and gene expression microarray methods will be employed to determine the profile of PAFc targeted genes. To explain the roles of PAFc in hepatome cells, related proteins will be conditional overexpressed or silenced in SK-Hep1 cells. RNA interference, protein pull-down and chromatin immunoprecipitation analysis will be employed to bridge PAFc functions with its mechanisms. In conclusion, we will use work of art cell models to elucidate profiles of target genes, interactome and functions of PAF complex, in order to link PAFc roles with it's molecular mechanisms in heptoma cells.

RNA聚合酶相关因子复合物（PAFc）在多种肿瘤细胞中发生突变或扩增，PAFc通过与MLL-AF9蛋白相互作用参与人类白血病的发生，我们的前期工作证明PAFc作为组蛋白精氨酸甲基化修饰的效应蛋白参与雌激素受体靶基因的活化，但其在肝癌细胞中的功能及机制还不清楚。在肝癌细胞中研究PAFc相互作用蛋白及其调控的靶基因，对阐明PAFc在肝癌中的功能和机制具有重要的意义。我们拟建立FlagPaf1替换内源Paf1表达的肝癌细胞系，利用Flag免疫共沉淀方法从该细胞系中鉴定PAFc相互作用蛋白组;通过ChIP-seq及基因芯片的方法在全基因组范围研究PAFc靶基因;在此基础上，在诱导型过表达及沉默特定蛋白的细胞模型中，利用定量PCR、Western Blot和染色体免疫共沉淀等方法将PAFc、相互作用蛋白、调控靶基因及其功能联系起来,阐明PAFc在肝癌细胞中的功能及其机制，为临床诊疗提供潜在的靶分子。