miR-29c和PDGFR-β的正反馈作用在介导慢性移植物血管病中的机制的研究

Chronic allograft vasculopathy (CAV) is always an important cause of the failure of organ transplantation. The up-regulation of PDGFR-β is a key factor in affecting the CAV development. In the previous study we found the over-expression of miR-29c could lead to PDGFR-β inhibition in Hela cell line, and the expression level of miR-29c was significantly different between CAV samples in rat cardiac allograft transplant models compared to the isograft transplantation. The bioinformatic results about the target genes of miR-29c indicated the relation between this microRNA molecule and CAV. It has also been verified in the hepatic stellate cells that miR-29 plays a role in the liver fibrosis by regulating the nuclear factor kappa B (NF-κB), an important transcription factor in the downstream of PDGFR. Based on the above results, we hypothesize that the positive feedback-loop between miR-29c and PDGFR-β boosting the CAV process. In other word, miR-29c was down-regulated after the activation of PDGFR-β, followed by the diminished inhibition of PDGFR-β expression.. In this study we intend to adopt vascular smooth muscle cell (VSMC) as the research object in vitro. The first, we should explicit the molecular mechanism of the transcriptional regulation of miR-29c by blocking the PDGFR-β downstream signaling pathway respectively. The second, we expect to define the target genes of miR-29c in VSMC through the dual luciferase reporter assay and western blotting combined with the infection of lentivirus particles, which were packaged with the pMIF-cGFP-Zeo vector or the anti-sense miRZip-29c vector. The third, the routine laboratory tests, including the MTT assay, the wound healing test and the Masson staining method, will be employed to evaluate the VSMC status after miR-29c is dysregulated. Finally, the experiment in vivo will be carried out in the rat abdominal aorta allograft transplantation model, which reflects the pivotal role of miR-29c by means of the positive feedback with PDGFR-β. . We anticipate this series of experiments will reveal the new molecular mechanism of CAV and serve as a foundation for the clinical development of therapeutic target.

CAV是器官移植失败的重要原因，PDGFR-β表达上调是影响CAV发展的重要因素。我们在实验中发现miR-29c对PDGFR-β的表达有抑制作用，且在发生CAV的大鼠心脏移植物中表达水平降低，其预测的靶基因中包括多个与CAV相关的分子。而PDGFR下游重要转录因子也被证实调控miR-29的转录。据此提出PDGFR-β活化后低调miR-29c，相应减弱对PDGFR-β的表达抑制形成正反馈环路加速CAV进程的假说。拟以VSMC为研究对象，分别阻断PDGFR-β下游信号通路，明确其调控miR-29c的分子机制；荧光素酶报告基因检测、免疫印迹等方法结合慢病毒感染过表达或抑制表达miR-29c，验证miR-29c的靶基因及调控miR-29c的转录因子；在细胞和动物模型水平验证miR-29c和PDGFR-β的正反馈在CAV病程中的关键作用。这将有利于深入理解CAV的分子机制并为临床诊疗方案提供新思路。

慢性移植肾肾病（chronic allograft nephropathy，CAN）是造成移植肾衰竭的主要原因，肾间质纤维化是CAN主要病理特征之一。miR-29c是我们在前期实验中发现的可能与CAN相关的microRNA分子，本课题拟研究miR-29c与其预测的靶分子PDGFR-β相互作用对于CAN进程的影响。.研究结果主要包括三个部分。第一部分主要使用荧光素酶活性测定实验和实时荧光定量PCR等方法验证了miR-29c对pdgfrb、cx3cl1等靶基因存在直接调控作用；而在体外培养的细胞中加入PDGF刺激后，包括miR-29c在内的miR-29家族分子的水平在波动。性变化72h后出现了明确的降低趋势。在体外实验中证实了miR-29c和PDGFR-β之间存在相互作用。研究miR-29c作用下靶基因的表达变化以及细胞因子作用对于miR-29的影响都是对miR-29c分子的生物学作用研究的重要方面。第二部分记录了在小鼠单侧输尿管结扎（Unilateral ureteral obstruction, UUO）致肾间质纤维化模型中观察到的miR-29及其靶分子的变化,用实时定量PCR和免疫组化等方法证实随着UUO术后肾脏纤维化的进展,miR-29分子的表达降低,并且伴随靶分子CX3CL1和PDGFR-β在RNA和/或蛋白水平的表达升高。动物模型水平观察到肾脏纤维化发生时，miR-29与靶分子之间此消彼长，佐证了miR-29在肾间质纤维化中的重要作用。第三部分集中描述了我们为研究miR-29分子构建的几个工具载体，包括过表达miR-29c的慢病毒载体,肾脏特异性表达miR-29c的载体,以及miR-29c的成像载体。几种工具载体的构建不仅有利于实现miR-29在体内和体外的过表达,以及肾脏组织的特异性表达,还利用Tet可诱导系统，实现只需要使用一种荧光素酶，且荧光素酶的表达与microRNA的表达正相关的，更为准确、方便的miR-29c的成像。这些载体是我们在研究过程中为了完成研究计划，解决出现的难点并对课题更深入研究的产物，将是我们研究miR-29c作用机制的重要工具。.上述结果在一定程度上证实了miR-29c在CAN进程中的重要作用，我们对其分子机制进行了初步探讨，并尝试用分子生物学手段弥补研究计划中的不足，以利于进一步研究miR-29c分子在CAN发生和进展中的作用。