LncRNA-C2dat2促进脑缺血再灌注后CaMKⅡ磷酸化激活P38信号通路诱导神经元凋亡的功能及机制研究

The mechanism of neuronal apoptosis after cerebral ischemia is not clear yet. Our previous study reveal that lncRNA C2dat2 overlap with the CaMK2D gene sequence which is the subtype of CaMKII, upregulated trend during cerebral ischemia reperfusion. The phosphorylation of CaMKII is decreased and p38 induced neuronal apoptosis is inhibited by silencing C2dat2. To our knowledge，transient cerebral ischemia and reperfusion can increase the level of CaMKII phosphorylation, and the phosphorylation of CaMKII is one of the important mechanisms for neuronal damage. Therefore, we hypothesis that long non-coding RNA C2dat2 promotes CaMKII phosphorylation to induced neuronal apoptosis through the p38 signaling pathway following cerebral ischemia. On the base of our preliminary studies，this project will further intend to verify that C2dat2 promotes CaMKII phosphorylation to induced neuronal apoptosis through the p38 signaling pathway following cerebral ischemia in vitro and in vivo. and then investigate the regulatory mechanism of C2dat2 to induce neuronal apoptosis following cerebral ischemia. This project will enrich the biological function of lncRNAs and the mechanism of neuronal apoptosis after cerebral ischemia in the lncRNAs level, and may provide a new target and new idea to benefit the prognosis and treatment of ischemic stroke.

脑缺血诱发神经元凋亡，但其调节的分子机制尚不完全清楚。本课题前期首次发现脑缺血再灌注诱导与CaMKⅡ亚型CaMK2D有序列重叠的lncRNA-C2dat2在缺血再灌注后缺血半影区表达上调。沉默C2dat2降低CaMKⅡ磷酸化，抑制p38诱导的神经元凋亡。已知CaMKⅡ磷酸化是神经元损伤的重要机制之一。因此我们提出假说：脑缺血再灌注通过诱导C2dat2的表达促进CaMKⅡ磷酸化进而激活p38信号通路而诱导神经元凋亡。本课题拟在前期研究基础上：1.通过体外细胞和体内动物实验验证脑缺血再灌注通过诱导C2dat2的表达，激活CaMKⅡ-p38信号通路而促进神经元凋亡；2.阐明脑缺血后C2dat2调控神经元凋亡的分子机制。本研究可以丰富lncRNA的生物学功能，在lncRNA水平揭示脑缺血再灌注神经元凋亡的分子机制，为寻找脑卒中治疗靶点提供新思路。

脑缺血-再灌注损伤（CIRI）是缺血性中风的重要病理生理过程，与各种生理和病理过程相关，包括自噬和凋亡。本课题通过体内和体外实验研究了CAMK2D相关转录本2（lncRNA-C2dat2）在脑缺血再灌注过程中的功能和作用机制。研究发现CIRI诱导C2dat2上调，上调的C2dat2促进神经元自噬和凋亡。机制上，C2dat2充当ceRNA负调控miR-30d-5p表达。miR-30d-5p靶向DDIT4的3'UTR，沉默DDIT4。另外，C2dat2与HSP70/HSP90的结合阻断了RNA诱导的沉默复合物装配，抑制了miR-30d-5p对其靶mRNA DDIT4沉默。体外N2a细胞OGD/R模型中，敲低C2dat2抑制DDIT4和Beclin-1以及LC3B II/I比值上调，p-mTOR/mTOR，P62和p-P70S6K/P70S6K比值的下调。这项研究首先揭示了C2dat2/miR-30d-5p/DDIT4/mTOR信号轴促进CIRI诱导的细胞自噬和凋亡，有助于更好地理解CIRI的机制并丰富CIRI中ceRNA假说。