MicroRNA-1298调控GJA1影响下肢动脉硬化闭塞平滑肌细胞行为研究

Excessive artery smooth muscle cell (ASMCs) proliferation and migration play pivotal steps in the genesis and process of arteriosclerosis obliterans (ASO). MicroRNA is recognized as an important governor of ASMCs behavior by regulating target gene expression at post-transcriptional level. Recently, we reported the microRNA expression profile in ASO samples using microRNA real-time PCR array screening. Among the most dysregulated microRNAs, miR-1298 was found downregulated in ASO media dramatically. Further introducing miR-1298 into ASMCs using miR-1298 mimic transfection significantly attenuated mitogens triggered ASMCs proliferation. It indicates that loss of miR-1298 expression might be related with ASO. However, the exact role and underlying mechanisms of miR-1298 regulating ASMCs behavior in ASO remains unknown. .Other than microRNAs, disordered intercellular communication between vascular cells is also an important initiator to promote ASMCs proliferation and migration. Gap junction is the predominant intercellular communication manner between ASMCs, mainly consists of Gap Junction protein Alpha-1 (GJA1). GJA1 upregulation was found in stenotic artery disease, and deletion of GJA1 was proved to be efficient in preventing artery restenosis in animal models. Our data also showed increased GJA1 protein expression in ASO samples. However, the reason for GJA1 upregulation in ASO remains unclear. .The inconsistence of GJA1 protein and mRNA expression in artery stenosis was reported, suggesting post-transcriptional regulation of GJA1 expression in ASO. In silico analysis revealed that miR-1298 could bind the GJA1 mRNA 3'UTR seed sequence and regulate GJA1 expression at post-transcriptional level. While the underlying mechanism remains unclear and deserves to be further studied..In the present study, we intend to use miR-1298 mimic and inhibitor transfection, Edu incorporation and transwell assay to determine the role of miR-1298 in regulating ASMCs behavior. Evaluate the therapeutic role of miR-1298 in treating artery stenosis by using lentivirus mediated overexpression in animal models. Use dual-luciferase assay to achieve the critical evidence of miR-1298 directly binds GJA1 mRNA 3'-UTR. Use lentivirus mediated GJA1 overexpression to determine the GJA1 involvement into miR-1298 conducted ASMCs behavior changing. And use in-situ hybridization and immunofluorescence dual staining to determine the correlation between miR-1298 and GJA1 in ASO samples..The present study would shed light on the possible role and mechanism of miR-1298/GJA1 in modulating ASMCs behavior in ASO. And provide more sufficient evidence for making miR-1298/GJA1 a new potential therapeutic approach for treating and preventing ASO.

动脉平滑肌细胞（ASMCs）增殖和迁移失控是导致下肢动脉硬化闭塞（ASO）的主要原因，microRNA在调控ASMCs行为变化中扮演重要角色，但是ASO中表达失调microRNA的功能仍不清楚。我们发现miR-1298在ASO中表达下调明显，升高miR-1298水平显著抑制ASMCs增殖，然而miR-1298对ASMCs迁移、凋亡等的影响以及其中的具体调控机制仍然未知，进一步分析发现可能与转录后水平调控GJA1表达有关。本研究拟采用microRNA转染、双荧光素酶报告基因、慢病毒表达、siRNA、原位杂交-免疫荧光双染等方法，旨在获得miR-1298通过GJA1调控ASMCs行为变化的可靠证据，评价和证实其在ASO发病中的作用。本项目将揭示ASO发病中miR-1298/GJA1调控ASMCs生物学行为变化的新机制，为确立miR-1298/GJA1作为干预ASO的新靶点提供更充分的科学依据。

下肢动脉硬化闭塞（ASO）的发病率逐年上升，严重危害我国人民健康。动脉平滑肌细胞（ASMCs）增殖和迁移失控是导致ASO的主要原因，microRNA在其中扮演重要角色。我们既往研究发现ASO中miR-1298表达下调显著，但其在ASO疾病进程中的作用机制未知。.在本项目中，我们发现miR-1298主要表达于动脉的平滑肌层，并且在ASO动脉平滑肌层中表达水平明显下降，这提示miR-1298的水平变化主要对动脉壁中的平滑肌细胞（ASMCs）产生影响。在体外培养的ASMCs模型中发现，miR-1298的表达受DNA甲基化的调控，并进一步在ASO标本中证实，启动子DNA高甲基化是导致miR-1298表达水平下降的主要原因。. 在体外培养的ASMCs中进行了一系列的功能试验，发现升高miR-1298水平后，可明显减少处于DNA复制期（S期）的ASMCs数量，进而明显抑制细胞的增殖能力，这一作用并不是通过诱导ASMCs凋亡所实现，同时还发现升高miR-1298水平可显著抑制ASMCs迁移能力。缝隙连接蛋白GJA1（Cx43）的mRNA 3’UTR区域存在可以与miR-1298结合的位点，提示Cx43是miR-1298的靶基因。在ASO临床样本中，miR-1298的表达水平与Cx43的表达水平呈显著负相关。在体外培养的ASMCs中，升高miR-1298水平可显著抑制Cx43蛋白的表达；抑制miR-1298则可明显促进Cx43蛋白的表达，而miR-1298并不影响Cx43的mRNA水平，提示miR-1298对Cx43的调控可能在转录后水平完成。将通过双荧光素酶基因报告法证明，miR-1298可直接与Cx43的mRNA 3’UTR结合调控其表达。进一步通过回复实验证实，miR-1298对ASMCs的调控是通过靶向Cx43的表达而完成。在大鼠动物模型中发现，过表达miR-1298可以显著减轻动脉狭窄程度，而抑制miR-1298则显加重了动脉狭窄，这一过程是通过抑制Cx43蛋白表达、减少ASMCs增殖和迁移所实现，提示miR-1298可作为潜在的治疗靶点。. 本项目揭示了ASO发病中miR-1298/GJA1（Cx43）调控ASMCs生物学行为变化的新机制，为确立miR-1298/GJA1（Cx43）作为干预ASO的新靶点提供更充分的科学依据。