基于环状RNA-0029966调控microRNA-185-5p/STAT5探讨补肾固冲法诱导母胎免疫耐受治疗复发性自然流产的作用与机制

Recurrent spontaneous abortion (RSA) is a difficult problem need to be solved urgently in clinics. In our previous study, we found that the disequilibrium of DC’s differentiation regulated by STAT5 was involved in RSA, and this phenomenon could be revised by the method of Bushenguchong. Based on these findings and combined with the latest development of circle non-coding RNA epigenetic regulation, we performed preliminary experiment of the project for further exploring the underlying mechanisms. Interestingly, we found that microRNA-185-5p was down-regulated in the blood of RSA patients, which has the potential binding site with STAT5. Meanwhile, it was found that circRNA-0029966 was up-regulated in RSA patients and there was potential binding site between circRNA-0029966 and microRNA-185-5p. Moreover, experiment in vitro found that the the expression of circRNA-0029966 was down-regulated after the treatment with the method of Bushenguchong. Therefore, we hypothesize that circle non-coding RNA may play important role in RSA. We plan to perform the current study by using modern molecular biology techniques such as CRISPR/Cas9 to further explore the regulatory effect and mechanisms of Bushenguchong method on circRNA-0029966/microRNA-185-5p/STAT5 axis for inducing maternal-fetal immunotolerance. The expected findings of the study will demonstrated the pathogenesis of RSA from the aspect of epigenetic modification of non-coding RNA, enrich the scientific meaning of “kidney governing reproduction” in TCM, and offer novel target and strategy for clinics.

复发性自然流产(RSA)是临床亟待解决的难题，我们在前期发现STAT5调控树突状细胞(DC)分化失衡打破母胎免疫耐受参与RSA发生并能被补肾固冲法逆转的基础上，结合环状RNA表观遗传前沿进展，通过预实验发现与STAT5具有潜在结合位点的microRNA-185-5p在RSA患者表达显著降低，而与microRNA-185-5p具有潜在结合位点的circRNA-0029966表达显著升高，并发现补肾固冲法能体外降低circRNA-0029966表达，推测环状RNA表观遗传调控发挥了重要作用。本项目拟采用CRISPR/Cas9等技术，探讨补肾固冲法调控circRNA-0029966/microRNA-185-5p/STAT5干预DC分化诱导母胎免疫耐受治疗RSA的作用与关键机制，以期从非编码RNA表观遗传角度阐释RSA发病机理，丰富中医学“肾主生殖”理论科学内涵，为治疗提供新的靶点。

复发性自然流产（RSA）发病率约为育龄妇女的3%～5%，约占全部妊娠的10%～15%，严重影响妇女生殖健康，其防治是临床亟待解决的难题。补肾固冲安胎历史悠久，临床反复证实了其有效性，但其调控机制尚未完全阐明。本项目在国家自然科学基金面上项目“基于环状RNA-0029966调控microRNA-185-5p/STAT5探讨补肾固冲法诱导母胎免疫耐受治疗复发性自然流产的作用与机制（81873337）”资助下，重点探讨了非编码RNA调控母胎免疫耐受在URSA中的作用及补肾固冲法靶向干预机制。研究内容及创新点包括：1.RSA患者及模型小鼠母胎界面pDC比例降低、cDC比例升高、pDC/cDC比率降低，circRNA/miRNA/STAT信号轴调控DC亚群偏移参与了RSA发生。2.补肾固冲法靶向调控circRNA及miRNA调控STAT3及STATA5信号通路，进而逆转DC亚群失衡，重塑母胎免疫耐受转态，治疗RSA。3.miRNA-103与STAT1通过碱基互补直接结合进而调控其表达，miRNA-103/STAT1信号通路活化促进M1型巨噬细胞极化参与了RSA发生，靶向miRNA/STAT信号轴调控巨噬细胞极化与功能可能成为重塑母胎免疫耐受治疗RSA的有效途径。4.补肾固冲法能显著降低RSA患者及模型小鼠母胎界面miRNA-103水平，降低STAT1表达并抑制M1型巨噬细胞极化，进而诱导母胎免疫耐受治疗RSA。5.非编码RNA调控的细胞自噬参与了RSA发生过程，表观遗传调控机制可能在此过程中发挥了重要作用，其深入探讨有望进一步揭示RSA发病机制，丰富中医学“肾主生殖理论”的科学内涵。. 研究进一步从表观遗传非编码RNA免疫调控角度阐释了RSA发病机理，丰富了中医学补肾固冲安胎理论的科学内涵。发表SCI论文7篇、中文核心期刊论文4篇；授权国家发明专利4项。培养青年岐黄学者、泰山学者青年专家各1人次，硕士研究生6人；研究成果获得中华中医药学会科学技术二等奖及山东省高等学校科学技术二等奖各1项，并继续得到了国家自然科学基金面上项目、山东省自然科学基金面上等资助，后续工作将标注基金号并及时向基金委汇报。