MED12/EGFR通路调控卵巢癌休眠和复发的机制研究

The high recurrence rate and chemoresistance of ovarian cancer are attributed to the emergence of tumor dormancy cells that are refractory to chemotherapy and clonally develop into recurrent tumors. The mechanisms underlying tumor dormancy have been elusive and not well characterized. In preliminary study, our group found that loss of MED12 could down-regulate EGFR to induce tumor dormancy in ovarian cancer cells. In this study, we will examine the expression level and mutation of MED12 by immunohistochemistry and gene sequencing to investigate the clinical significance of MED12 in ovarian cancer. CRISPR-Cas9 and transfection will be used to study the function of MED12 in tumor dormancy and chemoresistance of ovarian cancer in vitro. Xenografts in nude mice will be used to study the function of MED12 in tumor dormancy in vivo. Identification of downstream target genes of MED12 will be performed by microarray analysis and ChIP-seq assay. The mechanism of regulation of EGFR and downstream signaling by MED12 will be studied by ChIP, luciferase and other techniques. The study aims to investigate the effects of MED12 on tumor dormancy and its clinical significance in human ovarian cancer, to illustrate the function and mechanism of MED12 in regulating tumor dormancy, and to provide new strategies to overcome recurrence of ovarian cancer.

肿瘤休眠细胞的存在是卵巢癌化疗耐药及复发的主要原因，其机制仍未阐明。我们的前期研究发现，沉默MED12能下调EGFR的表达，诱导卵巢癌细胞进入休眠状态。本研究拟采用免疫组化及基因测序技术检测卵巢癌标本中MED12的表达水平及基因突变状态，分析MED12的表达水平及突变状态与卵巢癌化疗效果及复发的关系；以CRISPR-Cas9或基因转染技术沉默或过表达MED12，研究其对卵巢癌细胞休眠和耐药的影响；通过裸鼠移植瘤模型，研究MED12在体内调控卵巢癌休眠的作用；通过mRNA芯片及ChIP-seq等方法，探索MED12调控的候选靶基因；以ChIP、双荧光素酶报告基因等分子生物学方法，研究MED12调控EGFR及其下游信号通路的分子机理。本研究将揭示MED12在卵巢癌中的表达、突变及调控规律，阐明MED12调控卵巢癌休眠的作用及其分子机理，为卵巢癌治疗提供新思路，为克服卵巢癌耐药、复发提供新策略。

肿瘤休眠细胞的存在是卵巢癌化疗耐药及复发的主要原因，其机制仍未阐明。我们的前期 研究发现，沉默MED12能下调EGFR的表达，诱导卵巢癌细胞进入休眠状态。本研究采用免疫组化技术检测卵巢癌标本中MED12的表达水平状态，分析MED12的表达水平与卵巢癌化疗效果及复发的关系，结果发现MED12低表达与化疗耐药相关；以CRISPR-Cas9技术沉默MED12及慢病毒转染技术在MED12敲除细胞中恢复MED12的表达，研究其对卵巢癌细胞休眠和耐药的影响，结果发现MED12敲除后卵巢癌细胞体内外增殖能力降低，卵巢癌细胞进入休眠状态；通过mRNA芯片等方法，探索MED12调控的候选靶基因其下游信号通路的分子机理，结果发现MED12通过与EGFR的启动子相结合激活其转录，从而调控卵巢癌细胞的休眠状态；通过肿瘤-淋巴细胞共培养系统、免疫因子、趋化因子检测等实验研究 MED12 对卵巢癌细胞免疫功能的影响，结果发现MED12可能通过STAT1/ CXCL10调控卵巢癌细胞对免疫细胞的趋化能力从而影响其免疫功能。本研究揭示MED12在卵巢癌中的表达规律，阐明MED12调控卵巢癌休眠的作用及其分子机理，为卵巢癌治疗提供新思路，为克服卵巢癌耐药、复发提供新策略。