转移调控因子GATA4表观调控卵巢癌转移相关基因的机制及靶向阻遏研究

90% patients of high grade serous ovarian cystadenocarcinoma died due to metastasis, therefore, study the mechanism of ovarian carcinoma metastasis has always been a hot topic in cancer reaserch and never stopped. By means of ex vivo selection between the parental primary/metastatic tumors or several rounds of in vivo selection of metastasis in immunodeficiency mice models, researchers could find dozens or even hundreds of genes by these high-throughput gene expression based microarray analysis. However, identification and verification the functions of the single gene needed not only hard work, but also difficulty to show efficacy. More importantly, this approach ignored some genes itself played a role in the onset of tumor and also in metastasis, which was recently challenged by the proposal that metastasis may be directly driven by oncogenic mutations that exist in most cells of a primary tumor. Therefore, it raised the question that is it possible to find the upstream regulatory factors which could regulate a group of ovarian cancer metastasis related genes? In previous 2 years hard work, we successfully established the clinical specimens based histone methylation sequecing platform. After chromation immuno-precipitation sequencing (ChIP-seq) combined with RNA sequencing(RNA-seq), the huge amount of data were processed by our ingeneous bioinformatics data alalysis technique including motif-regulator analysis, prognostic analysis and cross-platform database analysis, etc. Finally, GATA4 was come out and proved to meet with all the criteria. Therefore, in the preliminary unpublished data, we hypothesize that understanding the function and regulation of “genomic organizer” GATA4 to ovarian cancer metastatis related genes, will shed valuable new light on the nature of histone methylation and will delineate currently unexplored new directions in drug design and cancer therapy.Genuinely, when the results mentioned above is incorporated with the novel oncolysis adenovirus, which combined the characteristics of oncolysis with targeted gene amplication and constructed by ourself previously, tumor-selective replication of the E1A mutant adenovirus would activate the native adenovirus E3 promoters to express the GATA4 cDNA preferentially in tumor cells and amplify the GATA4 protein. Thus, the aim of this project is beginning from the unknown function of GATA4 in histone methylation, and from the novel angle of the relationship between the histone methylation and cancer metastasis, to further explore the mechanism of how to regulate the downstream genes and the signal pathway by GATA4, by set up the new technique of ChIP-seq/RNA-seq and adenovirus construction technique. We believe this targeting GATA4 gene therapy of ovarian cancer metastasis will shed the new light on cancer recurrence and metastasis, span the key technique barrier in adenovirus gene therapy and ultimately provide a new strategy to prevent the recurrence and metastasis of cancer.

90%的高级别浆液性卵巢癌患者死于转移，因此，多年来卵巢癌转移的机制研究方兴未艾。前期研究中，课题组从表观遗传学角度，筛选能够调控一群卵巢癌转移相关基因的上游调控因子的思路，成功地建立了临床级组蛋白甲基化测序标本制备平台和调控关联性分析、预后分析、数据库跨平台分析等独创的生物信息学数据分析技术，从组蛋白甲基化与肿瘤转移的独特视角，以卵巢癌转移抑制调控因子GATA4为切入点，以困扰临床治疗实际的卵巢癌残留/转移灶为突破口，深入探讨GATA4 基因，通过募集去甲基化酶，抑制H3K4位点的甲基化而下调转移相关基因和抑制转移相关通路，来发挥其作为“Genomic Organizer”表观调控的机制，并引入选择性复制溶瘤病毒构建技术，从应用角度出发探索一条有效清除卵巢癌残留/转移病灶的靶向基因治疗新途径，从而达到探索肿瘤转移治疗新方法、解决腺病毒基因治疗关键技术障碍，最终实现靶向遏制肿瘤转移的目的。

课题组通过前期建立的临床级组蛋白甲基化测序标本制备和数据分析平台，初步推测GATA4与卵巢癌中基因的H3K4me3修饰密切相关，进而在已有的数据库中分析发现GATA4 在卵巢癌转移灶表达显著降低，且GATA4低表达与卵巢癌的预后差呈正相关；进一步应用慢病毒构建技术建立稳定转染GATA4高低表达的卵巢癌细胞系、Transwell侵袭和迁移体外试验以及卵巢癌原位移植瘤动物模型体内实验等一系列方法验证了GATA4抑制卵巢癌转移的生物学效应。应用GATA4抗体及H3K4me3抗体的ChIP-seq发现GATA4的靶基因与H3K4位点甲基化程度降低的基因有显著相关性；而将同步RNA-seq数据中得到的GATA4调控的差异基因、ChIP-seq数据中得到的GATA4的靶基因以及在H3K4me3位点存在差异的基因，这三者取交集分析共得到10个基因，提示它们可能是GATA4通过表观重编程机制参与调控卵巢癌转移的重要媒介。将这10个基因逐一进行ChIP-PCR、ChIP-reChIP、real time RT-PCR和western-blot验证后，明确了GATA4通过调控肿瘤转移相关基因GNG7及ADAM19的H3K4me3水平，从而调节GNG7及ADAM19基因表达来抑制卵巢癌转移的表观调控机制。最后，我们从治疗角度验证了应用MSCs运载以GATA4为靶点的选择性复制溶瘤病毒至肿瘤局部，能够有效清除卵巢癌病灶，从而为探索一种具有普遍方法学意义的肿瘤残留/转移治疗提供了新途径，同时也提升了腺病毒基因治疗的临床应用价值。