lncRNA与miRNAs相互作用调控Rap1A在同型半胱氨酸致免疫炎症的机制研究

Immuneinflammatory through the whole pathogenic process of homocysteine (Hcy). Previous studies have shown that miRNAs involved in the pathogenic regulation of Hcy, but the mechanism is not clear. LncRNA as a new regulatory factor, it has the consistency with miRNAs in the structure, has similarity in the regulating mode, so the interaction between the two becomes the focus of research. Rap1A is an important gene which regulates immuneinflammatory response, so it can be assumed that lncRNA interact with miRNAs to regulate Rap1A is an important mechanism in Hcy-induced immune and inflammatory. In order to verify this hypothesis, using gene chip to screen and identify that LNC00657 and miR-144 are the specific lncRNA and miRNAs in Hcy-induced immuneinflammatory. Using immune coprecipitation and biotin specific probe respectively precipitation or drop-down the compounds combined with miR-144 and LNC00657, clear the combination relations. Construct miR-144 and LNC00657 biosensor luciferase report plasmid, and with both expression plasmid and/or transfection respectively, analyze the change of the Rap1A, reveal the mechanisms of the interaction between the two to regulate immune inflammation induced by Rap1A, which provides the experimental basis for the prevention and treatment of Hcy.

免疫炎症反应贯穿于同型半胱氨酸(Hcy)致病的全过程，前期研究表明miRNAs是Hcy致病的重要机制，但其受何种因素及如何调控仍未清楚。lncRNA作为新调控因子与miRNAs在结构上具有一致性，在调节方式上具有相似性，因此探讨两者相互作用方式成为焦点；而Rap1A是调控免疫炎症的重要基因，故推测：lncRNA与miRNAs通过“海绵吸附”作用调控Rap1A是Hcy致免疫炎症的重要机制。为了验证该假说，运用基因芯片筛选并确定LNC00657与miR-144是Hcy致免疫炎症的特异性lncRNA和miRNAs；采用免疫共沉淀和生物素特异性探针分别沉淀或下拉与miR-144和LNC00657结合的复合物，明确两者结合关系；构建miR-144生物传感器荧光素酶报告质粒，并与两者过表达质粒和/或共转染，分析Rap1A的变化，揭示两者相互作用调控Rap1A致免疫炎症的机制，为防治Hcy提供实验依据。

高同型半胱氨酸血症(HHcy)可引起炎症性疾病，长链非编码rna (lncRNA)和microrna (miRNAs)均参与了这一生物学过程，但HHcy引起炎症的机制尚不清楚。我们使用lncRNA芯片发现，在Hcy处理的巨噬细胞和来自高蛋氨酸饮食的半胱硫氨酸β-合酶杂合敲除(CBS+/−)小鼠的外周血单核细胞中，lncRNA TGFB3-AS1高度表达。将编码TGFB3-AS1的腺病毒载体注射到CBS+/-小鼠中，进一步证实TGFB3-AS1在体内、体外通过Rap1a/wnt信号通路加速了hcy诱导的巨噬细胞炎症反应。同时，TGFB3-AS1与Rap1a相互作用，通过抑制其泛素化，在Hcy处理的巨噬细胞中减少Rap1a的降解。Rap1a作为miR-144的直接靶点，介导Hcy诱导巨噬细胞的炎症反应。生物信息学和RNA免疫沉淀(RIP)分析发现，TGFB3-AS1通过与pri-miR-144结合调控miR-144，并抑制miR-144的成熟，从而调控Rap1a的表达。更重要的是，我们收集了HHcy患者，发现高表达的TGFB3-AS1与健康人及HHcy患者血清中Hcy及促炎细胞因子水平呈正相关。这些结果揭示了TGFB3-AS1通过抑制miR-144成熟促进巨噬细胞炎症，从而缓解miR-144介导的功能性靶点Rap1a表达抑制的新机制。