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、血管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。肺血管重构的部分特征是显著的内侧和内膜肥大，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中胞质Ca2+ ([Ca2+]cyt)的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K+ （Kv)通道表达的下调和Kv电流（/K(V)）的降低有助于a）通过膜去极化提高[Ca2+]cyt来增加PASMC的增殖；b)通过抑制凋亡体积的减少和维持细胞质中足够的K+来抑制caspase和核酸酶来减少PASMC的凋亡。我们的数据显示，与正常的PASMC相比，IPAH-PASMC中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）是一种短rna，它与靶mRNA的3'-非翻译区（3'-UTR）互补序列结合，抑制mRNA翻译和/或诱导mRNA降解，从而抑制特定mRNA靶标的表达。因此，mirna是转录后调节剂，可能调节IPAH-PASMC中Kv通道mrna的水平。我们最近发现了几个mirna（例如miR-29b， miR- 138和miR-222）高表达，而几个Kv通道（例如KCNA1-7/10）在IPAH-PASMC中与正常PASMC相比显着下调。在正常PASMC中过表达miR-29b、miR-138或miR- 222会降低全细胞/K(V)，而抑制miR-29b则会挽救（即显著提高）IPAH-PASMC中的/K(V)。这些数据表明，miR-138、miR-222和miR-29b足以降低正常PASMC中的/K(V)，而miR-29b是IPAH-PASMC中降低/K(V)所必需的。基于这些观察结果，我们假设选择性上调的mirna参与了IPAH-PASMC中Kv通道的转录后抑制，mirna介导的Kv通道抑制有助于IPAH-PASMC中增殖增加和凋亡减少。为了验证这一假设，我们提出了三个具体目标：1)确定IPAH-PASMC中Kv通道的上调mirna和下调mirna，并确定IPAH-PASMC中哪些Kv通道亚基受到上调mirna的转录后调控；2)确定IPAH-PASMC中mirna的上调是否调控Kv通道靶mrna的稳定性和翻译；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确定IPAH- PASMC中Kv通道抑制的机制，并探索靶向miRNA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.