miR-23a抑制肺微血管内皮细胞凋亡及在脓毒症急性肺损伤中的作用

Sepsis-indued acute lung injury (ALI) and acute distress syndrome (ARDS) are inflammatory disorders of the lung and are associated with high rates of morbidity and mortality. Apoptosis of pulmonary microvascular endothelial cells (PMVECs) contributes to the impairment of the alveolar-capillary membrane and leads to alveolar flooding with serum proteins and edema fluid. Our previous work found that miR-23a may attenuate TNF-α-induced endothelial cell apoptosis through down-regulation of the caspase-7 and serine/threonine kinase 4-caspase-3 pathways. Thus, we hypothesize that miR-23a may be involved in the development of ALI. We will first up-regulate (mimics) or down-regulate (inhibitor) endothelial miR-23a expression and evaluate the role of miR-23a in the lipopolysaccharides (LPS）-induced apoptosis of PMVECs and EC permeability in vitro. To inhibit miR-23a in vivo, we will design single-stranded RNA oligonucleotides complementary to miR-23a (antagomirs). To up-regulate miR-23a in vivo, we will design cholesterylated miR-23a mimics (agomirs). After successfully delivery of miR-23a antagomirs/miR-23a agomirs into mice, the regulation role of miR-23a in the development of ALI will be studied in vivo. We next sought to determine the effect of miR-23a on expression of the potential target genes. Our findings will suggest an important role of miR-23a in the etiology of PMVEC apoptosis and serve as novel therapeutic targets for sepsis-induced ALI.

脓毒症急性肺损伤(ALI)是危重病患者死亡的主要原因。肺微血管内皮细胞（PMVEC）凋亡引起呼吸膜通透性增高，导致肺水肿、肺不张是ALI的病理基础。课题组前期工作证实了miR-23a通过在转录后水平抑制靶基因caspase-7和STK4的表达，减少脐静脉内皮细胞凋亡，提示miR-23a可能是脓毒症ALI中PMVEC凋亡调控的新靶点。本研究拟在体外培养的PMVEC中特异地沉默和过表达miR-23a，明确miR-23a对脂多糖诱导的小鼠PMVEC凋亡及对内皮细胞单层通透性的调控作用；并在miR-23a敲除和过表达的转基因小鼠体内进一步证实其对脓毒症时PMVEC凋亡及ALI的影响；最后在细胞模型，通过观察miR-23a对靶基因的作用，阐明其作用机制。本课题的实施不仅有助于从转录后水平揭示PMVEC凋亡的基因调控机制，也将为miR-23a作为脓毒症肺保护的新靶点提供坚实的理论基础和潜在的应用前景。

脓毒症急性肺损伤(Acute lung injury, ALI)是危重病患者死亡的主要原因，肺微血管内皮细胞（pulmonary microvascular endothelial cells, PMVEC）凋亡引起呼吸膜通透性增高，导致肺水肿、微小肺不张是ALI的病理基础。课题组前期工作证实了miR-23a通过在转录后水平抑制靶基因caspase-7和STK4的表达，减少脐静脉内皮细胞凋亡，提示miR-23a可能是脓毒症ALI调控的新靶点。我们首先成功建立了小鼠脓毒症盲肠结扎穿孔（CLP）模型，明确了肺脏是脓毒症中miR-23a作用的靶器官，通过HE、TUNEL和CD34抗体+cleaved-caspase-3免疫荧光双标实验以及CD34抗体+总的caspase-3l免疫荧光双标实验均证实进一步证实了脓毒症小鼠肺组织中miR-23a的表达含量显著减少并伴随有肺微血管内皮细胞凋亡增多，提示：miR-23a可能是ALI中肺血管内皮细胞凋亡调控的新靶点。接下来，本研究首先在在体培养的PMVEC中特异地沉默和过表达miR-23a，明确了过表达miR-23a对脂多糖 (lipopolysaccharides，LPS)诱导的小鼠PMVEC凋亡具有机制作用；而抑制miR-23a的表达能增加脂多糖 (lipopolysaccharides，LPS)诱导的小鼠PMVEC凋亡，随后通过生物信息学的方法，我们筛选出了miR-23a与凋亡相关的潜在靶基因包括有：Apaf1; Blcap; Bnip2; Bnip3l; Ccng1; Gja1; Gsk3b; Krit1; Map3k1; Nek6; Nuak2; Ppif; Ptk2b; Srpk2; Stat5b; Stk4; Tnfaip3; Tox3。随后利用Real-time PCR方法分别进行验证，得出TOX3和Map3k1符合microRNA转录后水平的反向调节原则。初步得出此基因为靶基因。接下来利用利用双荧光素酶报告实验，验证了TOX3和Map3k1确实是microRNA-23a的靶基因，阐明了其作用机制；最后在miR-23a敲除的和过表达的转基因小鼠体内进一步证实其对脓毒症时PMVEC凋亡的影响。本课题的实施不仅有助于从转录后水平揭示ALI时PMVEC凋亡的基因调控机制，也将为miR-23a作为脓毒症肺保护的新靶点提供坚实