研究目的：子宫内膜异位症相关卵巢癌（EAOC）的发病机制尚不清楚，目前认为其可能由子宫内膜异位症恶变而来。探索导致子宫内膜异位症发生恶性转化的关键调控基因具有重要的意义。观察SPEN基因在EAOC组织、卵巢子宫内膜样癌细胞系中的表达，并探讨SPEN在卵巢子宫内膜样癌细胞增殖和侵袭中的调控作用。

材料与方法：获得一例病理确诊为卵巢子宫内膜样癌合并不典型内异症的临床组织样本。显微激光分割技术用于同一患者已获得子宫在位内膜组织、异位卵巢内膜组织、不典型卵巢内异症组织和卵巢癌组织样本。分别提取DNA后，进行肿瘤高频突变基因芯片捕获高通量测序，寻找在不同样本中突变基因。选择卵巢癌样本中的特有突变基因SPEN进行进一步的分子机制研究，通过卵巢癌组织芯片联合免疫组化和实时荧光定量PCR技术分析SPEN在不同卵巢癌组织和细胞系中的表达。以人类卵巢子宫内膜样癌细胞系TOV-112D、IGROV1为主要研究目标,采用慢病毒感染技术沉默了SPEN表达,并通过平板克隆形成试验、EdU实验、Transwell试验、划痕实验,以及利用裸鼠皮下成瘤模型评价SPEN基因对细胞生长、侵袭、转化等功能方面的作用。通过二代基因组测序（RNA-seq）结合GO、KEGG分析SPEN差异表达前后细胞生物学功能的变化。应用SPEN免疫共沉淀联合质谱鉴定技术以及蛋白免疫印迹技术检测和验证SPEN下游蛋白的表达。

结果：肿瘤高频突变基因芯片捕获测序发现卵巢子宫内膜样癌的子宫在位内膜、卵巢异位内膜、卵巢不典型内异症、卵巢癌4种组织的共同突变为261个（包括BRCA2、EGFR、NF1、CTNN1B、KRAS、BRAF等），另有卵巢异位内膜、卵巢不典型内异症、卵巢癌的共同突变2个，卵巢不典型内异症和卵巢癌的共同突变49个（包括ARID1A、CHEK2、PTEN等），卵巢癌独有突变20个。其中卵巢癌独有突变SPEN基因在EAOC及以EAOC为主的混合型癌中表达升高（P＜0.01），在EAOC相关细胞系TOV-112D（P＜0.05）、TOV-21G（P＜0.01）和ES-2（P＜0.01）细胞样本中表达升高。使用慢病毒载体沉默SPEN后，TOV-112D及IGROV1细胞增殖能力、侵袭和迁移能力增加，增强裸鼠成瘤能力。SPEN基因表达被沉默后，RNA-seq转录组测序结合GO、KEGG分析，发现Notch信号通路激活，蛋白免疫印迹发现Notch信号通路相关下游蛋白DLL1、JAG1、 HES1、HEY1蛋白的表达明显增加。

结论：SPEN在EAOC中具有高表达，抑制SPEN的表达可以促进卵巢子宫内膜样癌的增殖和转移，激活Notch信号通路。SPEN有望成为EAOC预防和治疗的新靶点。

OBJECTIVE: Endometriosis associated ovarian cancer (EAOC) may come from endometriosis by malignant transformation manner. This research aimed to identify the key gene which triggers the malignant transformation of endometriosis to EAOC. We detected the SPEN genes expression in EAOC tumor tissues and ovarian endometrial cancer cell lines, and discussed the regulatory role of SPEN in ovarian endometrial cancer cell proliferation and invasion.

METHODS: There is 1 case of patient who underwent surgery in Peking Union Medical College Hospital (PUMCH) obstetrics and gynecology department. The pathology confirmed as ovarian endometrial carcinoma with atypical endometriosis. We presented high throughput DNA sequencing of tumor high frequency mutation gene chip to the paraffin embedded specimens of eutopic endometrium, ovarian ectopic endometrium, ovary atypical endometriosis and ovarian cancer tissues which were obtained by Laser-Capture Microdissection technique. In order to find the differentially expressed genes. Then we concentrated on SPEN gene. The expression of SPEN in different ovarian cancer tissues were analyzed by ovarian cancer tissue microarray combined with immunohistochemistry and different cell lines were analyzed by real-time fluorescence quantitative PCR. In this study, ovarian endometrioid cancer cell lines TOV-112D and IGROV1 were used for further research. SPEN expression was silenced by lentivirus infection technique, and the effects of SPEN gene on cell proliferation, invasion and migration were evaluated by colony formation assay, EdU assay, Transwell migration assay and subcutaneous tumor formation rate in mice. Analyzed the changes of cell biological function after SPEN silencing by high throughput RNA sequencing (RNA-seq) combined with GO and KEGG pathway analysis. We used co-immunoprecipitation combined with mass spectrometry and western blot to detect and verify the expression of downstream proteins of SPEN.

RESULT: High throughput DNA sequencing of tumor high frequency mutation gene chip result showed that the 4 specimens of eutopic endometrium, ovarian ectopic endometrium, ovary atypical endometriosis and ovarian cancer tissues have 261 same gene mutations (including BRCA2、EGFR、NF1、CTNN1B、KRAS、BRAF). There were 2 same mutations for ovarian ectopic endometrium, ovary atypical endometriosis and ovarian cancer tissues. 20 unique gene mutations were found in only ovarian cancer tissues. The expression of SPEN gene was increased in EAOC cancers (P < 0.01). It was also increased in EAOC related cell lines TOV-112D (P=0.014), TOV-21G (P < 0.01) and ES-2 (P < 0.01). Effective silencing of SPEN expression increased tumor cell proliferation, invasion and migration of TOV-112D and IGROV1 cells. After silencing SPEN, we used GO and KEGG analyzed RNA-seq data and found enrichment of Notch signaling pathway, and western blotting confirmed the over expressions of DLL1, JAG1, HES1 and HEY1 proteins.

CONCLUSION:. SPEN is highly expressed in EAOC, and the inhibition of SPEN expression can promote the proliferation and metastasis of ovarian endometrioid carcinoma and actives Notch signaling pathway. SPEN may be one of the mechanisms of ovarian endometrioid cancer and a new target for EAOC prevention and treatment.