老龄小鼠POCD模型大脑差异性circRNAs表达谱分析及功能研究

Postoperative cognitive dysfunction (POCD) is a serious neurological sequela after anesthesia and surgery and at risk of developing into Alzheimer's disease in the long term. However, the fundamental mechanisms of POCD remain indefinite and the sensitive biomarker for screening susceptible patients and the strategies for prevention and medication of POCD are scare. It is observed that circular RNAs (circRNAs) are involved in development of neurodegenerative diseases. Our preliminary work found that the expressions of some circRNAs dramatically changed in hippocampi of POCD model prior to the alteration of their corresponding mRNA and molecules related to synaptic activity. This study aims to sequence and screen whole-transcriptome (including mRNA and circRNAs) in vulnerable regions of brain in POCD model in aged mice. To harvest the different profiling of circRNAs in POCD, the preliminary screening is done according to the change of circRNAs enrichment in brain. The function of screened circRNAs is verified in primary cultured hippocampal neurons. The feasibility of screened circRNAs for biomarker of predicting POCD is verified in mice models of POCD. Summarily, all the designed experiments are conducted to screen the circRNAs as the biomarker of POCD and elucidate the role of circRNAs in the development of POCD and provide a new prospective for prevention and treatment of the POCD in aged population.

术后认知功能障碍（POCD）是麻醉手术后严重并发症，且存在远期向阿尔茨海默病转化的风险。但目前POCD的发病机制尚未阐明，不仅缺乏筛选高危人群的敏感性指标，而且无有效的预防和治疗措施。文献报道，circRNAs与神经退行性疾病的发生密切相关。项目组前期研究发现，POCD老龄小鼠海马部分circRNAs表达发生显著变化且早于其对应mRNA及突触功能相关分子表达变化。因此，本项目拟对老龄小鼠POCD模型多个易损伤脑区进行全转录组（circRNAs及mRNA）测序及筛选，继而进行差异性表达谱分析，然后对筛选出与POCD有关的circRNAs进行功能验证，最后在动物模型探讨筛选出的circRNAs作为预测POCD生物标记物及预防和治疗POCD及向阿尔茨海默病转化新靶点的可行性，该课题不仅有利于揭示老年病人POCD发病机制，而且为早期预防和治疗这种严重并发症的新策略提供理论依据。

课题组通过对16月龄老龄c57小鼠术后认知功能障碍（postoperative cognitive dysfunction, POCD）与非术后认知功能障碍(non-postoperative cognitive dysfunction, NPOCD)模型组，control组中认知功能障碍（cognitive dysfunction, CD）与非认知功能障碍（non-cognitive dysfunction, NCD）的小鼠进行海马和内侧前额叶皮质的转录组测序，分析各组间差异表达的circRNA、miRNA及mRNA。通过对组间差异表达的各RNA进行GO和KEGG功能富集分析，对变化显著和具有认知相关重要功能的RNA进行qPCR验证，从而筛选出参与POCD发生机制circRNA、miRNA和mRNA。由于circRNA稳定的环状结构，可作为POCD的诊断标志物和治疗靶点。为了进一步探究表达的circRNA、miRNA和mRNA在POCD发生中的可能作用，我们进行了circRNA-miRNA、miRNA-mRNA的结合位点预测，并通过双荧光素酶报告、质粒转染、荧光定量聚合酶链反应和Western blot验证得到了在POCD中产生交互作用的circRNA-miRNA和miRNA-mRNA，比如circAKT3与miR-106a-5p、circAsh1l与miR-106a-5p、miR-106a-5p可结合HDAC4等,为未来的临床和基础研究提供更多的诊断和治疗靶点。此外，circRNA除了对miRNA吸附作用之外，还具有调节亲本基因转录、调控蛋白表达和翻译功能，这些功能值得后续进一步发掘。同时，本课题组研究发现，已发表文献对于circRNA功能的探究主要集中于中小分子量的circRNA。对较大分子量（＞5000bp）circRNA的功能研究可能会受到病毒包被的限制，而绝大部分circRNA仍然是非编码RNA,这些大分子量circRNA同样值得深入探究其功能。

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