TGF-β信号转导通路对早期卵巢癌潜在生物标志物的影响及作用机制

Ovarian cancer (OC) is the most fatal gynaecological malignancy with a mortality rate of 70%. It is highly lethal because it tends to have progressed to an advanced stage upon diagnosis. The low survival rate results in part from (1) an inability to detect the disease at an early, curable stage, (2) the lack of effective treatment for the advanced cancer, and (3) our incomplete understanding of how the OC develops. Women carriers of a BRCA1 germ-line mutation have a cumulative lifetime risk of 40-50% for OC development. Previous study showed that BRCA1 mutated ovarian surface epithelial (OSE) cells resemble OC cells in showing several similarly dysregulated genes. In order to identify differentially expressed proteins in cells predisposed to OC, we subjected normal and BRCA1 mutated immortalized OSE (IOSE-b1) cells to MudPIT proteomics. We identified 319 differentially expressed proteins between BRCA1 mutated IOSE-b1 and control IOSE cell lines. By comparing to the HUPO plasma proteome and urine proteome datasets using the ProteinCenter bioinformatics software, we narrowed down the list to 109 secreted proteins. Among these proteins, 15 (8 upregulated and 7 downregulated) are present in biological fluids including plasma and urine, 47 (15 upregulated and 32 downregulated) are exclusively present in plasma and the other 47 (24 upregulated and 23 downregulated) are present only in urine. Thus, we hypothesize that BRCA1 mutated OSE cells exhibit molecular signatures and altered TGF-β signaling pathways associated with increased OC susceptibility that define early molecular events in disease and persist during tumorigenesis and some of upregulated proteins in these cells can therefore characterize OC biomarkers. The objectives of this study are (1) to validate the differentially expressed proteins, identified by MudPIT analysis, in immortalized and primary cultures of OSE derived from BRCA1 mutation carriers and control women and to determine the distribution of the potential biomarkers in human ovarian cancer tissues and body fluids; (2) to explore the role of TGF-β signaling pathway in regulating these proteins in cells predisposed to ovarian cancer. We will use independent methods such as immunoprecipitation and Western blot to validate proteins and further confirm them in tissue and body fluid samples. We will use molecular biology approach to investigate the mechanisms of the potentially altered TGF-β signaling pathways in cells. This work will help us to identify candidate biomarkers for the early detection of ovarian cancer and provide insights into the mechanisms of tumorigenesis. Our studies may lead to novel diagnostic markers and therapeutic targets for the prevention or treatment of this disastrous disease.

卵巢癌是死亡率最高的妇科肿瘤，可达70%。由于在早期可治愈阶段缺乏有效的检测手段，使得卵巢癌的5年生存率低下。携带BRCA1基因突变的妇女，发生卵巢癌的累积风险高达40-50%。研究表明，BRCA1突变的卵巢表面上皮细胞非常类似于卵巢癌细胞，在基因表达上有相似之处。用MudPIT蛋白质组学法，我们已发现在BRCA1突变的卵巢表面上皮细胞与正常细胞之间有319种差异表达的蛋白质，其中109种蛋白质属分泌性蛋白。因此我们假设，BRCA1 突变的卵巢表面上皮细胞已有特定的基因和蛋白质表达，并在疾病发生的早期已改变，且受TGF-β调节，而某些上调的分泌性蛋白可成为诊断早期卵巢癌的生物标记物。本课题目标是证实这些差异表达的蛋白质在卵巢癌细胞、组织和体液中存在，并探索TGF-β信号转导通路对这些差异表达蛋白的影响。这项研究将有助于我们发现检测早期卵巢癌的生物标记物，了解卵巢肿瘤的发生机制。

本课题主要研究TGF-β信号转导通路对早期卵巢癌潜在生物标志物的影响及作用机制。我们首先验证卵巢癌组织与正常卵巢组织之间的差异表达蛋白，重点探讨PIWI、CSTB、B2M、STAT1、PNPO、CMPK、FLOT1等蛋白作为卵巢癌潜在的生物标志物在人卵巢肿瘤中的表达及与临床病理特征的相关性。卵巢组织石蜡标本收集自2005年1月至2016年12月以来的卵巢肿瘤患者的卵巢（包括浆液性、黏液性和透明细胞性等卵巢肿瘤，分为良性肿瘤组、交界性肿瘤组和恶性肿瘤组）和无卵巢病变患者的卵巢或癌旁正常组织（对照组），用免疫组织化学染色法检测这些蛋白的表达。同时，用实时定量PCR和蛋白免疫印迹法检测部分新鲜组织中的mRNA和蛋白表达，并与病人的临床资料进行相关性分析。我们发现PIWI系列蛋白过度表达是卵巢癌三期伴有淋巴结转移患者的特征，而CSTB和B2M是卵巢肿瘤进展标志物，进一步研究发现PNPO与卵巢癌紫杉醇耐药相关。我们发现TGF-β1能调控上皮性卵巢癌OVCAR-3和SK-OV-3细胞内CSTB、B2M、PNPO、CMPK、STAT1等蛋白的表达，而且呈时间和剂量依赖性，TGF-βI型受体拮抗剂SB-431542能阻断TGF-β信号转导通路对这些蛋白的调控。在机制研究方面，我们把带有myc标签的STAT1和HA标签的TGF-βI型受体TβRI（有ALK1和ALK5两个亚型）或II型受体TβRⅡ共转染进入293T细胞，应用免疫共沉淀方法发现STAT1能与TGF-β受体结合，并相互影响彼此信号转导。利用双荧光素酶报告基因方法我们发现miR-143能与CSTB和PNPO miRNA上的3’-UTR相结合，进一步证实CSTB和PNPO是miR-143的靶基因，并且受TGF-β1调控。在功能研究方面，我们发现敲减这些基因能降低卵巢癌细胞增殖、迁移、侵袭能力和诱导细胞凋亡。我们用ELISA方法发现卵巢癌病人血清中FLOT1明显升高，结合CA125检查可明显提高卵巢癌检测的敏感性和特异性。综上所述，我们发现多个与卵巢肿瘤相关的生物标志物，部分在卵巢癌早期表达已有改变，而且受TGF-β信号转导通路调控。此外，干预这些生物标志物影响卵巢肿瘤细胞的生物学行为。这些成果能为治疗和预防卵巢癌提供新的理论依据，具有一定的科学意义和临床应用价值。