miRNA-139-5p通过靶向PDE4D调控cAMP/PKA/CREB信号通路抗抑郁的机制研究

Phosphodiesterase 4D (PDE4D) is the subtype of PDE4, which could firstly inhibit the signaling pathway in cAMP / PKA / CREB, then down-regulated expression of BDNF and reduced neurogenesis, finally induced depression occurs. From the previous research, we found that by inhibiting the protein PDE4D could activating the signaling pathway cAMP / PKA / CREB which can improve the incidence of depression. Also found miR-139-5p can target PDE4D, but the regulatory mechanism of this targeting relationship in depression has not been elucidated so far. Therefore, we hypothesized that miR-139-5p activates the cAMP/PKA/CREB signaling pathway by targeting PDE4D, up-regulating BDNF expression and promoting neurogenesis, and improving depression-like status. In order to define the theoretical basis of the pathogenesis of depression, and provide experimental evidence for the treatment of depression. This study is going to research the regulation mechanism of miR-139-5p on the development of depression base on based on the hypothesis miR-139-5p could target PDE4D, to elucidate the mechanism by which miR-139-5p regulates the signaling pathway of cAMP/PKA/CREB by targeting PDE4D.

磷酸二酯酶4D（PDE4D）是PDE4的一种亚型，可通过抑制cAMP/PKA/CREB 信号通路，下调BDNF表达和减少神经发生，诱导抑郁症发生。前期我们发现抑制PDE4D后可激活cAMP/PKA/CREB 信号通路，改善抑郁症。同时发现，miR-139-5p可靶向抑制PDE4D，但该靶向关系在抑郁症的调控机制尚未阐明。因此，我们提出假说：miR-139-5p通过靶向PDE4D激活cAMP/PKA/CREB 信号通路，上调BDNF表达和促进神经发生，可改善抑郁样状态。本课题拟基于miRNA-139-5p靶向PDE4D，研究miRNA-139-5p对抑郁症发生发展的调控机制，阐明miRNA-139-5p通过靶向PDE4D调控cAMP/PKA/CREB 信号通路抗抑郁的机制，为抑郁症的致病理论和临床药物靶点筛选提供实验证据。

本课题首先通过复制慢性不可预知温和应激（chronic unpredictable mild stress，CUMS）抑郁症小鼠模型分析海马组织PDE4D水平，采用Luciferase荧光素酶检测研究miRNA-139-5p/PDE4D的靶向作用，并利用原位杂交法分析miRNA-139-5p和PDE4D在细胞内的定位。然后，采用siRNA转染小鼠海马神经元HT-22细胞，通过qPCR和蛋白免疫印迹（western blot，WB），探索miR-139-5p靶向调控PDE4D mRNA的信号通路。最后，通过立体定位注射分别将腺相关病毒AAV- miR-139-5p和AAV- miR-139-5p sponge注入CUMS小鼠海马，采用酶联接免疫、WB和免疫荧光染色等方法，揭示miR-139-5p靶向调控PDE4D mRNA发挥抗抑郁作用的机制。研究结果显示：miR-139-5p可通过抑制PDE4D mRNA，激活cAMP/PKA/CREB/BDNF信号通路，上调BDNF表达水平，促进海马神经发生，发挥抗抑郁的作用。本课题证明了miR-139-5p是治疗抑郁症的潜在靶点，并揭示其通过靶向PDE4D激活cAMP/PKA /CREB/BDNF通路改善抑郁症的作用机制，为研制抗抑郁药提供了新靶点。项目资助发表SCI论文2篇，申请专利1项，培养博士后1人，博士研究生1人。项目投入经费20.50万元，支出20.50万元。