miR-153双靶向阻滞ROCK1/NFATc3途径抑制低氧诱导肺血管重构的机制研究

Pulmonary vascular remodeling is the key to hypoxia pulmonary hypertension. We have proved that ROCK1/NFATc3 pathway was involved in the formation of pulmonary vascular remodeling induced by hypoxia. The low expression of miR-153 was detected in pulmonary artery smooth muscle cells（PASMC）induced by hypoxia, which also showed reverse relationships with the expression of ROCK1 and NFATc3. We also found that the induction of miR-153 expression inhibits the proliferation of pulmonary artery smooth muscle cells and the expression of ROCK1 and NFATc3. Furthermore, we prove that miR-153 can bind to mRNA 3' UTR of NFATc3 and ROCK1. These results suggest that inhibiting the expression of ROCK1 and NFATc3 by miR-153 is the possible mechanism to inhibit pulmonary arterial remodeling induced by hypoxia. We will utilize recombinant expression vector to confirm the binding site between miR-153 and 3 'UTR of ROCK1 or NFATc3; analyze the correlation between hypoxia-induced pulmonary vascular remodeling regulated by miR-153 and targeted inhibiting of ROCK1 and NFATc3 expression in vitro and in vivo; clarify the regulation mechanism of miR-153 on hypoxia-induced pulmonary vascular remodeling. This research will provide preliminary results for understanding the pulmonary hypertension and pulmonary vascular remodeling induced by hypoxia.

肺血管重构是低氧性肺动脉高压发生的关键，申请者前期结果表明ROCK1/NFATc3途径参与调控低氧诱导的肺血管重构形成过程；在低氧诱导肺动脉平滑肌细胞中检测到miR-153表达下降，且与ROCK1和NFATc3表达负相关；在肺动脉平滑肌细胞中过表达miR-153可抑制低氧诱导的细胞增殖及ROCK1和NFATc3的表达；理论预测及荧光素酶报告基因结果证实miR-153可与ROCK1和NFATc3 mRNA的3’UTR靶向结合；提示miR-153抑制ROCK1和NFATc3表达是抑制低氧诱导肺动脉重构的可能机制。本项目拟利用重组表达载体验证miR-153与ROCK1和NFATc3的结合及位点，体内外分析miR-153靶向抑制ROCK1/NFATc3途径与其调控低氧诱导肺血管重构的相关性；明确miR-153调控低氧诱导肺血管重构的作用与机制。为深入研究低氧性肺动脉高压的机制及治疗靶点奠定基础。

第一部分 MiR-153通过靶向ROCK1 / NFATc3调节人肺动脉平滑肌细胞（HPASMCs）的增殖和迁移.本研究的目的是探讨microRNA（miR）-153通过作用于缺氧大鼠肺动脉平滑肌细胞ROCK1和NFATc3进而影响细胞增殖和迁移。低氧促进肺动脉的重构和肺动脉高压，6周低氧后，RVSP, RVHI, WA% and WT%显著升高（P<0.05）。低氧处理的HPASMCs，ROCK1和NFATc3基因表达升高，miR-153的基因表达降低。通过荧光素酶报告分析在293T细胞中miR-153靶向结合ROCK1和NFATc3。利用基因转染技术证明miR-153 mimic抑制ROCK1和NFATc3的蛋白表达，并进一步抑制缺氧条件下细胞的增殖和迁移。从而预测了miR-153是通过靶向缺氧条件下HPASMC的ROCK1和NFATc3而影响细胞的增殖和迁移。本研究在《Molecular Medicine Reports》发表SCI论文一篇，证明miR-153 可能同时靶向 ROCK1 和 NFATc3发挥双靶向阻滞 ROCK1/NFATc3 途抑制低氧诱导肺血管重构作用，为寻找新的治疗方案奠定理论与实验基础。..第二部分 β-arrestin1通过Akt/mTOR信号通路抑制缺氧损伤诱导的肺动脉内皮细胞自噬。.β-arrestin1是一种多功能胞质蛋白，作为PAH的重要保护因子，已引起广泛关注。然而，其在损伤肺动脉内皮细胞（PAECs）自噬中的作用仍有待确定。在这里，我们研究了β-arrestin1在缺氧应激下对人PAECs（hPAECs）自噬和凋亡的潜在影响。低氧刺激能增加自噬，降低hPAECs中的β-arrestin1水平。此外，缺氧暴露还可促进细胞增殖、迁移和抗凋亡能力。这些病理性改变在β-arrestin1过度表达后或用自噬抑制剂3-MA被逆转。β-arrestin1抑制缺氧性hPAECs的自噬和凋亡抵抗。低氧条件下，β-arrestin1上调Akt/mTOR信号通路的活性及降低BNIP3、Nix表达。本研究在《International Journal of Biochemistry and Cell Biology》发表SCI论文一篇，首次证明β-arrestin1通过激活缺氧性hPAECs中的Akt/mTOR途径来减少过度的自噬