Involvement of MicroRNA-133a in the Development of Arteriosclerosis Obliterans of the Lower Extremities via RhoA Targeting
Involvement of MicroRNA-133a in the Development of Arteriosclerosis Obliterans of the Lower Extremities via RhoA Targeting
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DOI：
10.5551/jat.27839
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发表时间：
2015-01-01
影响因子：
4.4
通讯作者：
Wang, Wenjian
中科院分区：
文献类型：
--
作者：
Li, Yongxin;Ouyang, Mao;Wang, Wenjian
目的：RhoA是动脉硬化闭塞症（ASO）患者动脉平滑肌细胞（ASMCs）增殖和迁移的重要调节因子。RhoA在心肌细胞和支气管平滑肌细胞中受microRNA-133 a（miR-133 a）调节。方法：采用原位杂交（ISH）和免疫组化（IHC）方法分别检测miR-133 a和RhoA在阿索临床标本中的定位。应用5-乙炔基-2 '-脱氧尿苷（EdU）、CCK-8、Transwell和伤口闭合试验检测人ASMC（HASMC）的增殖和迁移特性。采用实时荧光定量PCR（qRT-PCR）检测HASMCs中miR-133 a的表达，采用qRT-PCR和Western blotting检测RhoA的表达。miR-133 a在阿索组织和增殖的HASMCs中表达显著下调。相反，RhoA在阿索样品中上调。miR-133 a的下调可显著促进HASMCs的增殖和迁移，而miR-133 a的上调可显著抑制HASMCs的增殖和迁移。荧光素酶测定证实RhoA是miR-133 a的直接靶标。miR-133 a在HASMCs中的上调在蛋白水平上降低了RhoA的表达。然而，下调miR-133 a增加了RhoA蛋白的表达。结论：miR-133 a通过靶向RhoA调节HASMCs的功能，可能参与了阿索的发病过程。这些发现可能导致下肢阿索的潜在治疗靶点的开发。
Aim: RhoA is a critical factor in regulating the proliferation and migration of arterial smooth muscle cells (ASMCs) in patients with arteriosclerosis obliterans (ASO). RhoA is modulated by microRNA-133a (miR-133a) in cardiac myocytes and bronchial smooth muscle cells. However, the relationship between miR-133a and RhoA with respect to the onset of ASO in the lower extremities is uncertain.Methods: We employed in situ hybridization (ISH) and immunohistochemistry (IHC) to detect the location of miR-133a and RhoA in ASO clinical samples, respectively. 5-ethynyl-2'-deoxyuridine (EdU), cell counting kit-8 (CCK-8), Transwell and wound closure assays were utilized to determine the features of human ASMC (HASMC) proliferation and migration. The expression of miR-133a in the HASMCs was assessed using quantitative real-time PCR (qRT-PCR), while that of RhoA was examined via qRT-PCR and Western blotting.Results: We found miR-133a and RhoA to be primarily located in the ASMCs of ASO. miR-133a was significantly downregulated in the ASO tissues and proliferating HASMCs. In contrast, RhoA was upregulated in the ASO samples. The proliferation and migration of HASMCs was markedly promoted by the downregulation of miR-133a and inhibited by the upregulation of miR-133a. The Luciferase assay confirmed that RhoA was a direct target of miR-133a. The upregulation of miR-133a in the HASMCs decreased the RhoA expression at the protein level. Inversely, the downregulation of miR-133a increased the RhoA protein expression. Of note, the overexpression of RhoA in the HASMCs attenuated the anti-proliferative and anti-migratory effects of miR-133a.Conclusions: Our data indicate that miR-133a regulates the functions of HASMCs by targeting RhoA and may be involved in the pathogenesis of ASO. These findings may lead to the development of potential therapeutic targets for ASO of the lower extremities.