缺氧诱导的miR-210靶向BARD1调控细胞周期参与子宫内膜异位症的发展

Our previous study found hypoxia was involved in the pathogenesis of endometriosis. To better understanding the underling molecular mechanism, small RNA sequencing was performed to compare microRNAs expression difference between hypoxia and normoxia treated cells. Following QPCR confirmation, miR-210 was found elevated by hypoxia. Follow cytometry analysis demonstrated that knockdown miR-210 in endometrial stromal cells (ESCs) followed by hypoxia treatment significant arrested cells at G2/M phase, comparing with control cells treated by hypoxia alone. Transcriptone sequencing indicated that miR-210 inhibited the expression of genes involved in regulation of cell cycle, cell division and microtubule movement, which was highly consist with our cell cycle results. BARD1 was one of the candidate targets of miR-210 that greatly attracted our attention. Validation by QPCR and Western blot, BARD1 was largely repressed by miR-210. Furthermore, miR-210 binding sequence was found in BARD1 3’-UTR by bioinformatics softwares of Targetscan, RNAhybrid and miRanda. BARD1 was well known to bind with BRCA1, increasing it protein stability. By activating cell cycle checkpoint, heterodimer of BRCA1 and BARD1 arrested cell cycle at G2/M phase and initiate DNA damage repair. Herein, we would like to apply PCR, Western blot, immunohistochemistry and endometriotic xenograft nude mouse model to prove our hypothesis: hypoxia induced miR-210 might involve in development of endometriosis by promoting BRCA1 protein degradation via targeting BARD1, wich might probably inactivate cell cycle checkpoint and help endometriotic cells survive in local hypoxia microenvironment

缺氧参与子宫内膜异位症发病过程。我们前期microRNA测序显示，缺氧诱导子宫内膜间质细胞（ESCs）miR-210表达。ESCs敲除miR-210后缺氧处理，细胞周期被阻滞在G2/M期，细胞生长也被抑制，而单独缺氧处理ESCs并未有相应作用。转录组测序显示，miR-210显著下调ESCs细胞周期分裂相关基因的表达。QPCR及Western Blot (WB)验证发现BARD1被miR-210显著抑制，且BARD1 3’-UTR有miR-210结合序列。BARD1可与BRCA1相互结合，增强BRCA1蛋白的稳定性，促进其发挥其细胞周期Checkpoint调控作用。我们将通过PCR、WB、免疫组化及裸鼠内异囊肿模型，验证缺氧诱导的miR-210是否通过靶向BARD1，促进BRCA1蛋白降解，导致细胞周期Checkpoint机制失效，使细胞得以克服缺氧环境继续生长，促进内异症的发展。

缺氧参与子宫内膜异位症发病过程。本项目前期microRNA测序显示，缺氧诱导子宫内膜间质细胞 (ESCs) miR-210表达。在本项目中，我们对ESCs敲除miR-210后缺氧处理，细胞周期被阻滞在G2/M期，细胞生长也被抑制，而单独缺氧处理ESCs并未有相应作用。另外我们还发现miR-210显著下调ESCs细胞周期分裂相关基因的表达，QPCR及Western Blot (WB)验证发现BARD1被miR-210显著抑制，且BARD1 3’-UTR有miR-210结合序列。我们通过体外敲降miR-210-3p，明确了miR-210-3p在缺氧培养下对ESCs和Ishikawa细胞增殖、周期进展的作用；同时，过表达miR-210-3p的慢病毒载体转染至人子宫内膜异位症在位子宫内膜永生化间质细胞系hEM15A，结合二代测序和生物信息学分析寻找miR-210-3p在内异症间质细胞中的靶基因，并进行后续的RT-qPCR、WB、双荧光素酶报告基因分析。证明了缺氧上调的miR-210-3p通过靶向抑制BARD1的表达，促进BRCA1蛋白的降解，使BRCA1/BARD1蛋白复合物无法发挥正常的DNA损伤应答及细胞周期检测点功能，内异症细胞逃逸氧化应激损伤、细胞周期持续进展、细胞在宫腔外不断复制增殖，从而促进内异症的发展。另外，我们还发现体内敲降miR-210-3p或给予抗氧化剂维生素C补充均能有效抑制内异症小鼠异位囊肿的生长，这为维生素C作为子宫内膜异位症的辅助治疗提供了证据。综上所述，本项目验证了缺氧诱导的miR-210通过靶向BARD1，促进BRCA1蛋白降解，导致细胞周期Checkpoint机制失效，使细胞得以克服缺氧环境继续生长，促进内异症的发展。.