circRNA-miRNA-p38MAPK通路调控网络对缺血性脑卒中及其中医证候的影响与机制研究

Non-coding RNA (ncRNA) may be involved in the etiology and pathogenesis of traditional Chinese medical (TCM) syndromes. However, no research on circRNA of TCM syndromes in ischemia stroke (IS) has been reported. In our preliminary studies, by using circRNA microarray chips and cytoplasm-nucleus separation, two IS-related circRNAs have been identified to be located in cytoplasm and a circRNA-miRNA-p38 MAPK signaling pathway regulatory network was constructed. It is hypothesized that the circRNA-miRNA-p38 MAPK pathway network plays an essential role in pathogenesis of IS and its TCM syndromes by competing endogenous RNA (ceRNA) mechanism. To verify the hypothesis, we initially identify the circRNAs which could be biomarkers for the diagnosis and prognosis of IS and its TCM syndromes. Then, the biological function of circRNAs and its influence on the molecules and pathway within the network in vivo and in vitro are explored. Ultimately, the ceRNA mechanism of the circRNA-miRNA-p38 MAPK pathway network involving in IS and its TCM syndromes is clarified through fluorescence in situ hybridization (FISH), RNA immunoprecipitation (RIP), RNA pull down, luciferase assays and other experiments. This study could provide biological basis of ncRNA for TCM precision differentiation and potential therapeutic targets for syndrome differentiation and treatment of IS.

非编码RNA可能参与中医证候病因病机，但有关缺血性脑卒中（IS）中医证候的circRNA研究尚未见报道。课题组前期研究：已通过circRNA芯片、核质分离等实验鉴别出2个定位于细胞质的IS相关circRNAs，构建了可能与IS相关的“circRNA-miRNA-p38 MAPK通路调控网络”。据此假设：该调控网络可能通过竞争性内源RNA（ceRNA）机制影响IS及其中医证候的发生发展。本项目拟先从人群水平鉴别出IS及其不同中医证候的诊断及预后circRNA生物标志物；再通过细胞和动物模型，研究circRNAs的生物学功能及其对该调控网络中分子及通路的影响；最后，运用FISH、RIP、RNA pull down、双荧光素酶报告基因等实验，阐明该调控网络通过ceRNA机制影响IS及其中医证候的作用机制。研究结果可为精准中医辨证提供非编码RNA层面的生物学依据，为IS辨证论治提供治疗靶点。

本项目首先通过病例对照研究，发现hsa_circRNA_0105035、circFOXP1和circUSP36外周血表达在缺血性中风（Ischemic stroke，IS）及其中医证候病例组与对照组之间差异表达，表明这3个circRNAs与IS及其中医证候发生相关。通过诊断试验发现这3个circRNAs表达水平对IS及其中医证候具有诊断价值，前瞻性队列研究发现circUSP36和circFOXP1表达水平与IS患者出院后3个月或12个月的神经功能恢复相关，从而鉴别出可作为IS及其中医证候的诊断和预后预测生物标志物的circRNAs。进一步阐释了circFOXP1和circUSP36的作用机制：circFOXP1增加氧糖剥夺/再灌注（OGD/R）后细胞活性，抑制细胞凋亡水平；降低小鼠暂时性大脑中动脉闭塞（tMCAO）后的脑梗死体积和神经凋亡信号，降低神经功能缺损，促进脑缺血后的运动功能恢复；揭示了circFOXP1通过结合STAT3蛋白并降低其泛素化水平，促进STAT3蛋白表达，最终抑制脑缺血后神经凋亡信号，减轻脑损伤的作用机制。另外，发现circUSP36增强OGD/R后的细胞活性和增殖能力，抑制细胞凋亡水平；减轻小鼠tMCAO后的脑梗死体积，降低神经功能缺损，促进脑缺血后的运动功能恢复；揭示了circUSP36通过结合miR-139-3p调控SMAD3的表达而促进抗凋亡蛋白Bcl2的表达，从而减轻缺血后脑损伤的作用机制。本项目研究成果可为IS及其中医证候的诊断和预后预测提供潜在的生物标志物，有利于提高临床上对IS的诊断和预后判断能力；还阐明了circFOXP1和circUSP36在IS的作用机制，为促进IS神经功能恢复的治疗策略提供新的思路，从而促进中风的二级预防和三级预防，降低中风疾病负担。本课题已发表SCI论文5篇、中文论文1篇；培养硕士研究生4名、博士研究生1名。