Molecular mechanisms of downregulated Kv channels in IPAH: Role of microRNA

项目摘要/摘要 特发性肺动脉高压是一种致命的进展性疾病。 主要影响女性。肺血管重塑、持续性血管收缩和 原位血栓形成是IPAH肺血管阻力升高的主要原因 病人。肺血管重塑的部分特征是显著的内侧和 由肺动脉平滑肌细胞(PASMC)增加引起的内膜肥厚 促进PASMC增殖，减少PASMC凋亡。PASMC胞浆钙([Ca]Cyt)升高是一种 2 2 肺血管收缩的主要触发因素和PASMC增殖的重要刺激因素。 电压门控K(Kv)通道表达下调和Kv电流降低(Ik(V)) A)通过膜去极化提高[Ca]Cyt，促进PASMC增殖 2. 和b)通过抑制凋亡体积的减少和维持来减少PASMC的凋亡 细胞质中有足够的钾来抑制半胱氨酸酶和核酸酶。我们的数据显示， Kv通道的表达和活性降低，增殖增强， 与正常PASMC相比，IPAH-PASMC的细胞凋亡受到抑制。微RNA(MiRNAs)是 与3‘-非翻译区(3’-UTR)互补序列结合的短RNA 靶向信使核糖核酸抑制信使核糖核酸翻译和/或诱导信使核糖核酸降解 特定信使核糖核酸靶点的表达。因此，miRNAs是转录后调节因子， 潜在地调节IPAH-PASMC中Kv通道mRNAs的水平。我们最近确认了 几种高表达的miRNAs(例如miR-29b、miR-138和miR-222)，而 在IPAH-PASMC中，几个Kv通道(例如，KCNA1-7/10)显著下调 与正常PASMC相比。正常组织中miR-29b、miR-138和miR-222的过度表达 PASMC降低全细胞IK(V)，而抑制miR-29b可挽救(即显著 增强)IPAH-PASMC中的IK(V)。这些数据表明miR-138、miR-222和miR-29b是 在正常的PASMC中足以减少IK(V)，而对于减少的IK(V)则需要miR-29b 在IPAH-PASMC。基于这些观察，我们假设有选择地上调了 MiRNAs参与IPAH-PASMC中Kv通道的转录后抑制 MiRNA介导的Kv通道抑制有助于促进增殖和降低 IPAH-PASMC细胞凋亡。提出了三个具体的目标来检验这一假设：1) 识别Ipah中上调的miRNAs和下调的Kv通道- PASMC，并确定哪些Kv通道亚基转录后受 在IPAH-PASMC中上调的miRNAs；2)确定是否上调了IPAH-PASMC中的miRNAs- PASMC调节靶Kv通道mRNAs的稳定性和翻译；以及3)确定 MiRNA介导的Kv通道转录后抑制在PASMC增殖中的作用 和细胞凋亡。这项研究的长期目标是确定 抑制IPAHPASMC中的Kv通道并探讨靶向miRNA的可能性 开发IPAH的治疗方法。

Project Summary/Abstract Idiopathic pulmonary arterial hypertension (IPAH) is a fatal and progressive disease that predominantly affects women. Pulmonary vascular remodeling, sustained vasoconstriction and in situ thrombosis are the major causes for the elevated pulmonary vascular resistance in IPAH patients. Pulmonary vascular remodeling is characterized in part by significant medial and intimal hypertrophy, due to increased pulmonary arterial smooth muscle cell (PASMC) proliferation and decreased PASMC apoptosis. A rise in cytosolic Ca ([Ca ]cyt) in PASMC is a 2+ 2+ major trigger for pulmonary vasoconstriction and an important stimulus for PASMC proliferation. Downregulation of voltage-gated K (Kv) channel expression and decrease in Kv currents (IK(V)) + contribute to a) increasing PASMC proliferation by raising [Ca ]cyt via membrane depolarization 2+ and b) decreasing PASMC apoptosis by inhibiting apoptotic volume decrease and maintaining sufficient K+ in the cytoplasm to inhibit caspases and nucleases. Our data showed that the expression and activity of Kv channels are reduced, while proliferation is enhanced and apoptosis is inhibited, in IPAH-PASMC compared to normal PASMC. MicroRNAs (miRNAs) are short RNAs that bind to complementary sequences in the 3'-untranslated regions (3'-UTR) of target mRNAs to inhibit mRNA translation and/or induce mRNA degradation, thereby inhibiting the expression of specific mRNA targets. miRNAs are thus posttranscriptional regulators that potentially regulate the level of Kv channel mRNAs in IPAH-PASMC. We recently identified several miRNAs (e.g., miR-29b, miR-138 and miR-222) that are highly expressed, whereas several Kv channels (e.g., KCNA1-7/10) are significantly downregulated, in IPAH-PASMC compared to normal PASMC. Overexpression of miR-29b, miR-138 or miR-222 in normal PASMC decreases whole-cell IK(V), whereas inhibition of miR-29b rescues (i.e., significantly enhances) IK(V) in IPAH-PASMC. These data suggest that miR-138, miR-222 and miR-29b are sufficient to decrease IK(V) in normal PASMC, while miR-29b is necessary for the decreased IK(V) in IPAH-PASMC. Based on these observations, we hypothesize that selectively upregulated miRNAs are involved in the posttranscriptional inhibition of Kv channels in IPAH-PASMC and the miRNA-mediated Kv channel inhibition contributes to increasing proliferation and decreasing apoptosis in IPAH-PASMC. Three Specific Aims are proposed to test this hypothesis: 1) To identify the miRNAs that are upregulated and the Kv channels that are downregulated in IPAH- PASMC, and to determine which Kv channel subunits are posttranscriptionally regulated by the upregulated miRNAs in IPAH-PASMC; 2) To determine whether upregulated miRNAs in IPAH- PASMC regulate the stability and translation of target Kv channel mRNAs; and 3) To determine the role of miRNA-mediated posttranscriptional inhibition of Kv channels in PASMC proliferation and apoptosis. The long-term goal of this study is to define the mechanism underlying the inhibition of Kv channels in IPAHPASMC and to explore the possibility to target miRNA for developing therapeutic approaches for IPAH.