Vof16参与调控Gnb3在糖尿病认知功能障碍中的作用及机制研究

Our previous studies found that a long noncoding RNA Vof16 highly expressed in hippocampus tissue of diabetic rat with cognitive dysfunction by lncRNA microarray. Furthermore, bioinformatic analysis suggested Gnb3 was its candidate target and Vof16 might function as a competing endogenous RNA to “sponge” miR-205 and modulate the concentration and biological function of Gnb3. Gnb3 gene showed highly expressed in hippocampus tissue of diabetic rat with cognitive dysfunction. Thus, we hypothesize that the mechanism of Vof16 “sponge” miR-205 and regulated Gnb3 may affect cognitive damage induced by diabetes. Base on the above research, we choose high glucose induced hippocampal neuron cells and diabetic cognitive dysfunction rats, and aim for the reliable evidence that the mechanism of Vof16 “sponge” miR-205 and regulated Gnb3 is involved in cognitive damage induced by diabetes by the technologies of molecular biological and morphological methods, and in vitro & in vivo. We hope to investigate the biological values and the molecular mechanisms of the mechanism of Vof16 “sponge” miR-205 and regulated Gnb3 in the development of diabetic cognitive dysfunction in more details. Moreover, the current project will provide new targets and theoretical basis for the diagnosis and treatment of diseases such as diabetes and cognitive dysfunction.

前期我们通过芯片筛选发现lncRNA Vof16是一条高表达于糖尿病认知功能障碍大鼠海马组织中的差异lncRNA，生物信息学的分析提示Gnb3可能为Vof16的调控靶标，且miR-205分别与Vof16、Gnb3有相同的应答元件，进一步体内实验验证Gnb3高表达于糖尿病认知功能障碍大鼠的海马组织。因此，我们分析，糖尿病时Vof16可能通过吸附miR-205，调节Gnb3的表达，进而参与了糖尿病认知功能损害。基于上述研究基础，本课题拟以高糖诱导的海马神经元细胞、糖尿病认知功能障碍大鼠为研究对象，借助体内外实验和分子生物学、形态学等技术和方法，旨在获得Vof16通过吸附miR-205调节Gnb3参与糖尿病认知功能损害的可靠证据，证实Vof16通过吸附miR-205调节Gnb3这一机制在糖尿病认知损害发生、发展中的生物学价值，为糖尿病、认知障碍等相关疾病诊治提供新的靶点和理论基础。

我们通过芯片筛选发现lncRNAvof16是一条高表达于糖尿病认知功能障碍大鼠海马组织中的差异lncRNA，生物信息学的分析提示Gnb3可能为Vof16的调控靶标，且miR-205分别与Vof16、Gnb3有相同的应答元件，进一步体内实验验证Gnb3高表达于糖尿病认知功能障碍大鼠的海马组织。在高糖培养的大鼠海马细胞中，双荧光素酶报告基因试验，分别证实了lncRNAvof16与miRNA-205，miRNA-205与Gnb3的靶向结合关系。CCK-8实验证实lncRNAvof16可调控miR-205对海马神经元细胞增殖的影响。Western Blot实验证实miR-205能在蛋白水平抑制Gnb3的表达。进一步构建lncRNAvof16过表达载体，将其注射于大鼠海马齿状回，72小时后行为学实验证实大鼠出现严重记忆损伤。脑片Nestin/NeuN双标染色显示：过表达lncRNAvof16组大鼠海马神经病变性、坏死明显，细胞周围间隙增大。Western Blot证实lncRNAvof16能在体内上调Gnb3的表达。综合上述实验结果，LncRNAvof16在糖尿病认知功能障碍大鼠的海马组织中高表达，并通过结合miR-205，干扰了miR205和靶基因Gnb3的结合，使得Gnb3在糖尿病认知功能障碍大鼠海马组织中高表达，进一步损害大鼠的学习记忆能力。我们进一步用过表达lncRNAvof16载体转染H19-7细胞，收集了上述细胞和对照组细胞，进行了高通量转录组测序和代谢组测序，整合多组学数据后，我们发现LncRNAvof16的过表达可影响海马神经元的自噬，lncRNAvof16可通过提高mTORC1底物S6K的磷酸化水平降低海马神经元的自噬，可能在糖尿病认知损害的发病机制中起重要作用。