lincRNA-p21/miR-181b-5p/PKC-δ环路调控帕金森病炎症机制的研究

The role of microglia mediated neuroinflammation in the progression of Parkinson's disease (PD) is well established, but the regulatory mechanism of microglial activation is still not clear. Many studies show that both lncRNA and miRNA are important regulatory factors in neurodegenerative disease and immune inflammation, our previous study showed that lincRNA-p21 served as a repressor in p53-dependent transcriptional responses, and promoted the activation of microglial cells, with the mutual regulation of miR-181b-5p and PKC-δ. Therefore, according to the "competing endogenous RNA(ceRNA)" theory put forward in recent years, this study intends to verify that lincRNA-p21 acts as a ceRNA, competitivly "sponges" miR-181b-5p, and indirectly up-regulates PKC-δ in molecular, cellular, and animal model levels. Moreover, as PKC-δ could promote the expression of p53, lincRNA-p21 forms a double-negative feedback loop with miR-181b-5p/PKC-δ and promotes the activation of microglia synergistically, which deteriorates the development of PD. We are also going to explore the correlation of lincRNA-p21 with the onset, grade, and drug efficacy of PD in clinical level. Our study might clarify the role of lincRNA-p21-mediated micraglial activation in the progression of PD, therefore contribute to the present knowledge of PD molecular biology, provide new target for biological intervention of PD, and explore quantitative index of the clinical diagnosis and the therapeutic evaluation of PD.

小胶质细胞介导的中枢炎症反应在帕金森病（PD）进展中起关键作用，然而小胶质细胞的活化机制尚不明确。LncRNA与miRNA是神经退行性病变和免疫炎症反应的重要调控因子，我们前期研究发现，lincRNA-p21作为p53的下游效应分子，与miR-181b-5p/PKC-δ相互作用，促进小胶质细胞的活化。因此，根据ceRNA理论，本课题拟从分子、细胞、动物模型水平证明lincRNA-p21作为ceRNA，竞争性“吸附”miR-181b-5p，间接上调PKC-δ，同时PKC-δ又促进p53/lincRNA-p21的表达，彼此形成一正反馈环路协同促进小胶质细胞的活化，加剧PD进展；同时我们将在临床水平对lincRNA-p21与PD发病、分级、药物疗效进行相关性分析。该研究不仅可以丰富现有对PD分子生物学机制的认识，为PD药物治疗提供新的靶点，也有望发掘PD临床诊断和疗效评价的量化指标。

背景：帕金森病（Parkinson’s Disease, PD）是最常见的慢性神经退行性运动障碍疾病之一。小胶质细胞介导的中枢炎症反应在PD进展中起关键作用，然而小胶质细胞的活化机制尚不明确.研究内容：1、分子水平：通过下调/过表达 p53/lincRNA-p21、PKC-δ、miR-181 家族，利用实时荧光定量 PCR 及蛋白免疫印迹技术，分析其在小胶质细胞静息及活化状.态中的表达及其中相互调控关系；进一步利用双荧光素酶报告实验、RIP 及 RNA pull down，确定 lincRNA-p21 作为 ceRNA，竞争性吸附并下调 miR-181 家族，调控 PKC-δ 的表达。2、细胞水平：下调/过表达 p53/lincRNA-p21、PKC-δ、miR-181 家族，利用蛋白免疫印迹技术及流式细胞技术检测小胶质细胞表型相关分子的变化，明确p53/lincRNA-p21/miR-181 家族/PKC-δ 环路在小胶质细胞活化中的作用；进一步利用流式细胞术研究 lincRNA-p21 对小胶质细胞神经毒性的影响。3、动物水平：构建 LPS 制备 PD 炎症模型、MPTP 制备 PD 急性模型，利用实时荧光定量 PCR、蛋白免疫印迹检测 p53/lincRNA-p21/miR-181 家族/PKC-δ 表达；立体定向注射下调/上调小鼠中脑黑质 lincRNA-p21 表达，利用实时荧光定量 PCR、蛋白免疫印迹、免疫荧光以及流式细胞术，检测 lincRNA-p21 对 PD中小胶质细胞的激活效应以及神经毒性作用。.重要结果、关键数据：1、p53 诱导 lincRNA-p21 表达，促进小胶质细胞活化，加剧其神经毒性作用。2、 miR-181 家族（miR-181a/b/c/d）靶向抑制 PKC-δ，抑制小胶质细胞活化。3、 lincRNA-p21 作为 ceRNA，竞争性“吸附”miR-181 家族，间接上调 PKC-δ，同时 PKC-δ 又促进 p53/lincRNA-p21 的表达，彼此形成一正反馈环路协同促进小胶质细胞的活化。.科学意义：本研究，明确了p53/lincRNA-p21/miR-181 家族/PKC-δ 环路协同促进小胶质细胞持续活化，加剧 PD 进展。