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)是一种致命的进行性疾病，主要影响女性。肺血管重构、持续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。肺血管重塑的部分特征是显著的中膜和内膜肥大，这是由于肺动脉平滑肌细胞(PASMC)增殖增加和PASMC凋亡减少所致。PASMC胞浆钙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)通道表达的下调和Kv电流的降低(/K(V))有助于a)通过膜去极化提高[Ca~(2+)]Cyt来促进PASMC的增殖；b)通过抑制凋亡体积的减少和维持胞浆中足够的K来抑制caspase和核酸酶，从而减少PASMC的凋亡。我们的数据显示，与正常的PASMC相比，IPAH-PASMC的Kv通道的表达和活性降低，增殖增强，细胞凋亡受到抑制。MicroRNAs(MiRNAs)是一种与靶mRNAs的3‘-非翻译区(3’-UTR)上的互补序列结合的短RNA，可抑制mRNAs的翻译和/或诱导mRNAs的降解，从而抑制特定mRNAs的表达。因此，miRNAs是转录后调节因子，可能调节IPAH-PASMC中Kv通道mRNAs的水平。我们最近发现了几个miRNAs(例如miR-29b、miR-138和miR-222)在IPAH-PASMC中高表达，而几个Kv通道(例如KCNA1-7/10)与正常PASMC相比显著下调。在正常的PASMC中，miR-29b、miR-138或miR-222的过表达降低了Ipah-PASMC的全细胞/K(V)，而抑制miR-29b则挽救(即显著增强)了IPAH-PASMC的全细胞/K(V)。提示在正常PASMC中，miR-138、miR-222和miR-29b足以降低/K(V)，而在IPAH-PASMC中，miR-29b是降低/K(V)所必需的。基于这些观察结果，我们推测选择性上调的miRNAs参与了IPAH-PASMC中Kv通道的转录后抑制，并且miRNA介导的Kv通道抑制有助于促进IPAH-PASMC的增殖和减少凋亡。为了验证这一假说，我们提出了三个特定的目标：1)确定IPAH-PASMC中上调的miRNAs和下调的Kv通道，并确定IPAH-PASMC中上调的miRNAs对哪些Kv通道亚单位进行转录后调控；2)确定IPAH-PASMC中上调的miRNAs是否调节靶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增殖和凋亡中的作用。本研究的长期目标是明确IPAH-PASMC中Kv通道抑制的机制，并探索靶向miRNA的可能性，以开发IPAH的治疗方法。

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.