miRNA靶向调控TLR7/MyD88/IRF5通路影响缺血性中风风痰瘀阻证发生发展的机制研究

Resent studies have shown that miRNA plays an important role in the development of syndrome of traditional Chinese medicine (TCM). The applicant’s preliminary experiments found that the mRNA expression of TLR7/MyD88/IRF5 pathway genes were associated with the occurrence of ischemic stroke (IS) with wind-phlegm blocking collaterals symptom. Additionally, we selected 11 miRNAs targeting this pathway through bioinformatics analysis. Therefore, hypothesis is as follows: these miRNAs may influenece the occurrence of IS with wind-phlegm blocking collaterals symptom through targeted regulating the TLR7/MyD88/IRF5 pathway. To verify the hypothesis, we firstly detect the miRNA expression of peripheral blood in IS patients with wind-phlegm blocking collaterals symptom and controls, which can identify miRNAs related to IS with wind-phlegm blocking collaterals symptom from the level of people. Then, the targeted binding of miRNAs with their target gene will be validated using dual-luciferase reporter gene assay. Finally, research on the influence of miRNAs in target gene transcription and translation will be conducted in the oxygen-glucose deprivation model applying plasmid construction, transfection, RT-PCR, Western Blot techniques, which clarify the mechanism from the cellular level that miRNA targeted regulate the TLR7/MyD88/IRF5 pathway and affect the development of IS with wind-phlegm blocking collaterals symptom. It can provide basic data for revealing the essence of syndromes of stroke, and can also provide molecular evidence for the objectification of TCM syndrome.

近期研究表明miRNA在中医证候的发生中起重要作用。申请人前期实验发现，TLR7/MyD88/IRF5通路基因mRNA表达与缺血性中风（IS）风痰瘀阻证发生相关；并通过生物信息学分析筛选出11个靶向该通路的miRNA。据此假设：这些miRNA可能通过靶向调控TLR7/MyD88/IRF5通路影响IS风痰瘀阻证的发生。为验证假设，我们拟先测定IS风痰瘀阻证患者、对照外周血miRNA表达，从人群水平鉴别出与IS风痰瘀阻证相关的miRNA。再运用双荧光素酶基因报告技术验证miRNA与靶基因的靶向结合。并采用氧糖剥夺细胞模型，运用质粒构建、转染、RT-PCR、Western Blot等技术，研究miRNA对靶基因转录和翻译的影响，从细胞水平阐明miRNA靶向调控TLR7/MyD88/IRF5通路影响IS风痰瘀阻证发生发展的作用机制。为揭示中风证候的实质提供基础数据，为中医证候的客观化提供分子依据。

缺血性脑卒中严重危害人们的健康，给社会带来了沉重的负担。有研究表明miRNA可能影响缺血性脑卒中中医证候的发生。本项目完成了以下研究：首先在50例缺血性中风风痰瘀阻患者和50例健康对照中，运用实时荧光定量PCR（qRT-PCR）测定了11个miRNAs的外周血表达水平，发现miR-19a、miR-148a、miR-342-3p、miR-320d、miR-548ac、miR-3135b、miR-3940-3p、miR-4429这8个miRNAs可能与缺血性中风风痰瘀阻证发生相关。然后，运用双荧光素酶报告基因实验，验证了miR-3940-3p与IRAK4靶向结合、miR-3135b与IRF5靶向结合。进一步采用人脑微血管内皮细胞（HBMEC）建立氧糖剥夺模型，通过细胞转染（慢病毒和mimic转染）、qRT-PCR、Western Blot （WB）、Enzyme-linked immunosorbent assay（ELISA）等实验技术，明确了miR-3940-3p影响IRAK4蛋白、炎性细胞因子IL-6和IL-8的表达。本项目发现了8个可能与缺血性中风风痰瘀阻证相关的miRNAs，这些miRNAs可能可以作为缺血性中风风痰瘀阻证的生物标志物。并进一步阐明了miR-3940-3p通过与IRAK4基因3'UTR区靶向结合负向调节IRAK4蛋白的表达，从而通过调控TLR7/MyD88/IRF5通路来影响炎性细胞因子IL-6和IL-8的表达，进而影响缺血性中风风痰瘀阻证的发生。本研究成果为中医证候客观化提供了分子生物学客观依据，为中医证候的分子机制研究提供了新的思路，为进一步实现中医精准医学打下基础，对于推动中医现代化发展有重要意义。本课题已发表论文4篇，培养4名硕士研究生。