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

描述（由申请人提供）：特发性肺动脉高压（IPAH）是一种主要影响女性的致死性进行性疾病。肺血管重构、持续性血管收缩和原位血栓形成是IPAH患者肺血管阻力升高的主要原因。肺血管重构的部分特征在于由于肺动脉平滑肌细胞（PASMC）增殖增加和PASMC凋亡减少而导致的显著的中膜和内膜肥大。PASMC胞浆Ca ~（2+）（[Ca ~（2+）]cyt）升高是肺血管收缩的主要触发因素，也是PASMC增殖的重要刺激因素。电压门控性K+（Kv）通道表达下调和Kv电流（/K（V））降低有助于a）通过膜去极化提高[Ca 2 +]cyt增加PASMC增殖和B）通过抑制凋亡体积减少和维持细胞质中足够的K+以抑制半胱天冬酶和核酸酶来减少PASMC凋亡。我们的数据表明，与正常PASMC相比，IPAH-PASMC中Kv通道的表达和活性降低，而增殖增强，凋亡抑制。微小RNA（miRNAs）是与靶mRNA的3 '-非翻译区（3'-UTR）中的互补序列结合以抑制mRNA翻译和/或诱导mRNA降解的短RNA，从而抑制特异性mRNA靶标的表达。因此，miRNA是转录后调节因子，其潜在地调节IPAH-PASMC中Kv通道mRNA的水平。我们最近鉴定了几种miRNA（例如，miR-29 b、miR- 138和miR-222），而几种Kv通道（例如，与正常PASMC相比，KCNA 1 -7/10）在IPAH-PASMC中显著下调。正常PASMC中miR-29 b、miR-138或miR- 222的过表达降低了全细胞/K（V），而miR-29 b的抑制挽救了（即，显著提高IPAH-PASMC的K（V）/K（V）。这些数据表明，miR-138、miR-222和miR-29 b足以降低正常PASMC中的K（V），而miR-29 b是IPAH-PASMC中降低K（V）所必需的。基于这些观察，我们假设选择性上调的miRNA参与IPAH-PASMC中Kv通道的转录后抑制，并且miRNA介导的Kv通道抑制有助于IPAH-PASMC中增殖的增加和凋亡的减少。本研究拟从以下三个方面来验证这一假说：1）鉴定IPAH-PASMC中上调的miRNAs和下调的Kv通道，并确定哪些Kv通道亚基受上调的miRNAs的转录后调节; 2）确定IPAH-PASMC中上调的miRNAs是否调节靶Kv通道mRNA的稳定性和翻译; 3）探讨miRNA介导的Kv通道转录后抑制在PASMC增殖和凋亡中的作用。本研究的长期目标是确定IPAH-PASMC中Kv通道抑制的潜在机制，并探索靶向miRNA用于开发IPAH治疗方法的可能性。

DESCRIPTION (provided by applicant): 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 Ca2+ ([Ca2+]cyt) in PASMC is a 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 (/K(V)) contribute to a) increasing PASMC proliferation by raising [Ca2+]cyt via membrane depolarization 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 /K(V), whereas inhibition of miR-29b rescues (i.e., significantly enhances) /K(V) in IPAH-PASMC. These data suggest that miR-138, miR-222 and miR-29b are sufficient to decrease /K(V) in normal PASMC, while miR-29b is necessary for the decreased /K(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 IPAH- PASMC and to explore the possibility to target miRNA for developing therapeutic approaches for IPAH.