LincR-PPP2R5C靶向调控ARNT/PP2A/STAT3和吸附miRNA介导Th2细胞分化参与支气管哮喘发生发展

Th2 cells are essential in the pathogenesis of allergic asthma. We used the next-generation sequencing, qRT-PCR and FISH in the hope to get novel long non-coding RNA in the CD4+T cells in the asthma. One of novel lncRNA, located in the cytoplasma, was LincR-PPP2R5C. We hypothesize that LincR-PPP2R5C may recruit transcription factor ARNT and suppress PPP2R5C/PP2A/STAT3 signal pathway. Meanwhile, LincR-PPP2R5C may de-repress the effects of ARNT on GATA-3, the master transcription factor of Th2, and therefore boost the differentiation of Th2 cells. Besides, LincR-PPP2R5C may sponge with miR-132-3p and miR-148a, which would suppress the differentiation of Th1 and Th17 cells. We aim to clarify the above hypothesis with the gain-of-function and loss-of-function strategies in vivo and in vitro.

哮喘是常见多发性疾病，Th2细胞在哮喘气道慢性炎症中发挥主要作用。我们应用高通量测序筛查、qRT-PCR以及FISH等方法，结合生物信息学分析，获得一条定位在胞浆中的新LncRNA，我们命名为LincR-PPP2R5C。在前期实验的基础上，申请者提出假说——（1）LincR-PPP2R5C招募ARNT，降低PPP2R5C转录，抑制PP2A/STAT3通路和Th2细胞分化；（2）LincR-PPP2R5C与ARNT结合，去除ARNT对GATA-3的抑制，促进Th2细胞分化；（3）吸附miR-132-3p和miR-148a抑制Th1/Th17细胞分化。本项目拟构建过表达或者沉默载体以及全敲除和CD4+T细胞条件性敲除小鼠，考察LincR-PPP2R5C在哮喘发生发展中介导CD4+T细胞分化的调控机制。上述研究有助于揭示LncRNA在Th2细胞相关疾病中的角色。

II型CD4+T辅助（Th2）细胞在支气管哮喘的发病机制中占主导地位，既往文献证实长非编码RNA（lncRNAs）参与调控免疫细胞分化，但目前对长非编码RNA在哮喘的Th2分化中的作用了解甚少。本项目发现了一种未经报道的lncRNA，lincR-PPP2R5C，是一种主要表达于细胞核中的基因间非编码RNA，其在哮喘小鼠脾CD4+T细胞和体外的诱导分化的Th2细胞中明显升高。初始CD4+T细胞过表达LincR-PPP2R5C后抑制了Th1的分化。相反，敲低lincR-PPP2R5C抑制了Th2的分化。ChIRP和FRET实验的结果表明，LincR-PPP2R5C和PPP2R5C启动子之间形成了RNA-DNA三聚体，通过顺式作用促进了相邻基因PPP2R5C的表达，PPP2R5C是磷酸酶PP2A的亚基之一，lincR-PPP2R5C过表达后通过PPP2R5C激活了PP2A引起Th1/Th2极化的改变。LincR-PPP2R5C全敲除和CD4条件性敲除的哮喘小鼠的气道高反应性低于对照哮喘小鼠，BALF中嗜酸性粒细胞、血清中IgE的总水平以及BALF中IL-4和IL-13的水平下降，肺中Th2细胞的比例降低，提示敲除和CD4细胞条件性敲除LincR-PPP2R5C后。本项目首次发现了lncRNA在哮喘中调节Th2细胞的具体证据，有助于理解 CD4+T 细胞分化发育过程，也有助于深入理解 Th2细胞介导的过敏性哮喘及其他相关疾病的发生机制。