Expression analysis on microfibrillar associated protein 5 (MFAP5) protein treated ovarian cancer cell line OVCA432

Ovarian cancer is the fifth most common form of cancer in women in the United States. Among different types of ovarian cancer, epithelial ovarian cancer is the most common and is highly lethal, however, prognostic and predictive markers, which can be used to predict chemoresponse and patient survival, have not been thoroughly explored. One critically important yet often overlooked component to the tumor progression process is the tumor microenvironment. Primarily composed of fibroblasts and extracellular matrix proteins (ECM) as well as endothelial cells and lymphocytic infiltrate, the tumor microenvironment has been shown to directly affect cell growth, migration, and differentiation through secreted proteins, cell-cell interactions and matrix remodeling (Tlsty and Coussens, 2006). The tumor microenvironment has the potential to promote tumor initiation of normal epithelial cells and facilitate progression of malignant cells, thereby, presenting a unique approach to diagnosing, understanding and treating cancer. Using a whole-genome oligonucleotide array platform to perform transcriptome profiling on the fibroblastic stromal component microdissected from a series of advanced stage high-grade serous ovarian adenocarcinomas, we identified a transcriptome signature for the ovarian cancer associated fibroblast (CAF). We further functionally characterized one of the identified genes, MFAP5, and we showed that stromal MFAP5 is a prognostic marker associated with poor patient survival. In addition to that, to investigate the signaling machanism and the effect of MFAP5 treatment on ovarian cancer cells, transcriptome profiling of MFAP5 treated OVCA432 high-grade serous ovarian cancer cells was performed. Further functional studies showed that stromal MFAP5 modulated ovarian cancer cell motility and invasion potential. High grade serous ovarian cancer cell line OVCA432 was used. Total RNA was isolated from control samples and MFAP5 treated cancer cell samples at 48 hours post-treatment. Followed by cDNA synthesis, IVT and biotin labeling, samples were then hybridized onto Affymetrix Human genome U133 plus 2.0 microarrays. For each treatment group, three independent samples were prepared for the microarray experiment.

卵巢癌是美国女性中第五大高发癌症类型。在各类卵巢癌亚型中，上皮性卵巢癌最为常见且致死率极高，然而可用于预测化疗应答与患者生存情况的预后及预测标志物，尚未得到充分探索。

肿瘤进展过程中一个至关重要却常被忽视的组成部分是肿瘤微环境。该微环境主要由成纤维细胞、细胞外基质蛋白（extracellular matrix, ECM）、内皮细胞以及淋巴细胞浸润物构成，已有研究证实其可通过分泌蛋白、细胞间相互作用及基质重塑直接影响细胞生长、迁移与分化（Tlsty与Coussens，2006）。肿瘤微环境可促进正常上皮细胞的肿瘤起始过程，并加速恶性细胞的进展，由此为癌症的诊断、机制研究与治疗提供了全新的切入点。

本研究采用全基因组寡核苷酸阵列平台，对一系列高级别晚期浆液性卵巢腺癌中经显微切割获取的成纤维性基质组分开展转录组表达谱分析，最终鉴定出卵巢癌相关成纤维细胞（cancer-associated fibroblast, CAF）的转录组特征。我们进一步对鉴定得到的基因MFAP5进行了功能表征，并证实基质MFAP5是与患者不良生存显著相关的预后标志物。

此外，为探究MFAP5的信号转导机制及其对卵巢癌细胞的作用，本研究对经MFAP5处理的高级别浆液性卵巢癌细胞系OVCA432进行了转录组表达谱分析。后续功能实验表明，基质MFAP5可调控卵巢癌细胞的迁移与侵袭潜能。本研究采用高级别浆液性卵巢癌细胞系OVCA432作为实验模型：于MFAP5处理后48小时，从对照组与处理组的癌细胞样本中提取总RNA；经互补DNA（complementary DNA, cDNA）合成、体外转录（in vitro transcription, IVT）与生物素标记后，将样本杂交至Affymetrix人类基因组U133 Plus 2.0微阵列芯片。每个处理组均设置3份独立生物学重复用于本次微阵列实验。