CircULK2-miR-29a-5p-PTEN通路参与调控血管性痴呆自噬的作用及分子机制研究

Our pre-experiments found that circULK2 was significantly up-regulated in the hippocampus of vascular dementia (VD) rats, and the qRT-PCR results were consistent with the sequencing results, and the expression of circULK2 was also significantly increased in the primary hippocampal neuron of OGD model. With overexpression of circULK2, miR-29a-5p expression was decreased significantly, PTEN and LC3-II protein expressions were increased. While knockdown circULK2, miR-29a-5p expression was significantly increased, PTEN and LC3-II levels were down-regulated. Based on this, we speculate that circULK2 can promote VD autophagy, but the mechanism is unclear. Bioinformatics analysis predicted that circULK2 can interact as a ceRNA with miR-29a-5p, and dual luciferase assay found that miR-29a-5p can target with 3'UTR of PTEN mRNA. Previous studies have shown that PTEN is a positive regulator of autophagy, so we speculate that circULK2 may act as a ceRNA to bind with miR-29a-5p, up-regulate the expression of PTEN, and promote VD autophagy. In this study, we used lentiviral vector RNA interference technology in vitro and in vivo to explore the mechanism of circULK2 regulating autophagy in VD, and provide a basis for seeking VD interventional targets.

预实验发现，circULK2在血管性痴呆(VD)大鼠海马中显著上调，qRT-PCR验证与测序一致，原代海马神经元OGD模型中circULK2表达也显著升高。过表达circULK2，miR-29a-5p表达显著下降，PTEN、LC3-II表达增加；而敲低circULK2，miR-29a-5p显著升高，PTEN、LC3-II水平下调。由此我们推测circULK2可促进VD自噬，但机制不明。生物信息学分析circULK2可作为ceRNA与miR-29a-5p互作，双荧光素酶检测miR-29a-5p能靶向结合PTEN mRNA 3’UTR。研究表明PTEN是自噬正调节分子，故我们又推测circULK2可能作为ceRNA与miR-29a-5p结合，上调PTEN表达，促进VD自噬。本课题从体外和在体水平，采用慢病毒载体RNAi技术，探讨circULK2调控VD自噬的机制，为寻求VD干预靶标提供依据。

血管性痴呆(vascular dementia, VD)作为公认的唯一可以防治的痴呆，若早期干预有可能逆转。但至今仍无特效干预手段，归因于VD发病机制尚不明确及缺乏可靠的生物学标志物，给临床早期诊断与治疗带来了极大的挑战。因此，寻找特异的生物学标志物及治疗VD新的基因靶标，对VD早期干预具有重要的意义。本项目以双侧颈总动脉永久结扎术制备的VD大鼠模型和原代海马神经元细胞氧糖剥夺损伤模型为研究对象，采用慢病毒载体介导的 RNAi技术，从体外和在体水平，通过荧光素酶报告基因检测、FISH、circRIP等技术，验证circULK2通过与PTEN-miR-29a-5p互作，在促进VD自噬中发挥生物学作用。本项研究将为VD干预靶标提供理论依据，对早期诊治VD起到积极的作用。