FOXF2与SMAD6共调控COL5A2转录表达参与宫腔粘连发病的机制研究

Intrauterine adhesions (IUA), a fibrosis disease, remain an urgently clinical challenge for seriously threatening to female reproductive health. Excessive deposition of collagen element seems to be a major pathological basis in IUA. However, the molecular mechanisms remain elusive. Our primary results were shown as follows: Both FOXF2 and COL5A2 were overexpressed in the endometrium from women with IUA and during the human endometrial stromal cell (HESC) fibrogenesis, while SMAD6 expression was decreased; There were potential binding sites of FOXF2 and SMAD6 in the promoter region or in the enhancer region of COL5A2. Thus, COL5A2-induced deposition of collagen regulated by highly expressed FOXF2 and (or) lowly expressed SMAD6 in HESC might play a important role in the molecular mechanisms of IUA formation. Based on the scientific questions aboved, a series of experiments are designed as: Correlation of FOXF2, SMAD6 and COL5A2 need to be validated with more clinical samples; To determine whether FOXF2 and SMAD6 could regulate fibrosis in vitro; To determinine whether FOXF2 could involve IUA formation in vivo; FOXF2 and (or) SMAD6 may bind to the promoter region or enhancer region of COL5A2 directly and promote transcriptional expression of COL5A2, and such mechanism needs to be validated by Luciferase experiment; The model of combinatorial transcriptional regulation of FOXF2 and SMAD6 to COL5A2 needs to be validated by .CO-IP and CHIP assay. The present study aims to elucidate one of the molecular mechanisms of IUA formation, which could provide a strong theoretical basis for future innovative treatment.

宫腔粘连（IUA）作为纤维化病变亟待解决，胶原过度沉积是其病理基础，但分子机制不明。预实验结果：15例临床样本芯片数据和子宫内膜间质细胞（HESC）验证实验显示FOXF2与COL5A2在IUA中高表达，SMAD6低表达；CHIP实验显示COL5A2启动子/增强子具有FOXF2和SMAD6的潜在结合位点。因此，HESC内高表达的FOXF2和（或）低表达的SMAD6调控COL5A2致胶原沉积可能是IUA形成的重要分子机制。本研究拟：扩大临床样本验证FOXF2、SMAD6与COL5A2相关性；体外验证FOXF2和（或）SMAD6调控纤维化；体内验证FOXF2和（或）SMAD6参与IUA；荧光素酶验证FOXF2和（或）SMAD6结合COL5A2启动子/增强子调控其转录；CO-IP和CHIP技术验证FOXF2与SMAD6在转录水平调控COL5A2的模式。阐明IUA分子机制，为探寻治疗新方向提供依据。

宫腔粘连（IUA）是严重危害女性生殖健康的疾病之一，其病理基础是子宫内膜基底层损伤后修复障碍，子宫内膜上皮被纤维组织替代，最终发生纤维化导致粘连形成。胶原过度沉积是IUA纤维化发生的关键，但关于胶原合成的调控机制目前尚不明确。Foxf2是叉头框转录因子家族的成员之一，在间质中特异表达，参与胶原的合成。课题组前期研究通过IUA患者子宫内膜组织进行全基因测序发现Foxf2、Smad6与COL5A2异常表达，且通过启动子分析发现COL5A2上游启动子区域可能存在Foxf2与Smad6的转录结合位点。而Smad6是TGF-β/骨成型蛋白（ BMP）信号通路的关键负调控分子，可以抑制胶原的合成。本研究拟通过体内外实验验证Foxf2和Smad6参与纤维化，并通过免疫共沉淀（CoIP）、染色体免疫共沉淀(ChIP)、荧光素酶报告基因实验验证Foxf2与Smad6共调控COL5A2的转录表达，阐明其在IUA纤维化中的发病机制，为IUA的防治提供理论依据。研究内容如下：1.下调Foxf2或上调Smad6表达对TGF-β1诱导的人子宫内膜间质细胞株（THESCs）纤维化的影响，2.Foxf2与Smad6共调控COL5A2的转录表达的作用机制，3.Foxf2与Smad6在大鼠IUA发病中的作用及其妊娠的影响。研究结果示：TGF-β1促进THESCs纤维化，纤维化标记物COL1A1、COL5A2、FN、a-SMA等表达明显增加；以及促进细胞增殖和细胞周期由G0/G1期进入S期；下调Foxf2或上调Smad6表达均可抑制TGF-β1诱导的纤维化改变，抑制细胞的增殖，阻滞细胞周期由G0/G1期向S期进入；其中同时下调Foxf2和上调Smad6表达效果更明显；2.COL5A2上游启动子区域存在Foxf2和Smad6共同的转录结合位点；3.下调Foxf2或上调Smad6表达均可减轻双重损伤法导致的大鼠IUA，减轻纤维化，改善子宫内膜腺体表达，抑制COL5A2及COL1A1的表达；增加αvβ3和胞饮突的表达，改善子宫内膜容受性，提高妊娠胚胎数量。研究提示Foxf2和Smad6均参与了THESCs纤维化过程，两者相互结合，在COL5A2启动子区域具有相同的转录结合位点，对IUA防治提供了重要的科学依据。