miR-431经TGF-β/ SMAD4通路调节肺发育中II细胞功能的机制研究

Neonatal lung development mechanism has not been fully understood. Previous research: miR-431 was a differentially expressed miR during lung development, highly expressed in lungs and serum of RDS patients, inhibited proliferation of AECII, and induced downregulation of SP-B; After the pre-test and analysis of bioinformation, SMAD4 is an improtant target gene, and the pathway involved inTGF-β was critical for lung development. This suggested miR-431 could regulate lung development and related diseases by TGF-β/SMAD4. This study planed to: (1)Dynamic observation at different time points of rat lung morphology and expression of miR-431 and TGF-β pathway; (2)Construction of miR-431 expression vectors and SMAD4 3'UTR luciferase reporter gene system, demonstrated the regulation of TGF-β pathway at different time points; (3)Construction of miR-431 expression lentivirus vectors, studied the proliferation, trans-differentiation, apoptosis of AECII and the synthesis and secretion of SPs on different time points. Because no studies of miR-431 involved in lung development were reported, it's possible to find new regulatory theory, and provide the theoretical basis for the common mechanism of certain lung developmental diseases.

新生儿肺发育机制至今未能完全明了。前期研究：miR-431为肺发育中筛选出的一条连续差异miRNA，同时高表达于RDS患儿血清及肺组织，抑制II型肺泡上皮（AECII）增殖、致SP-B表达下调；经预实验及信息学分析，SMAD4为其重要靶基因、SMAD4参与的TGF-β通路为肺发育重要通路，提示miR-431可借由TGF-β/SMAD4通路参与肺发育及相关疾病的调控。本研究拟：（1）动态观察不同时间点大鼠肺发育形态和miR-431、TGF-β通路连续表达变化；（2）构建miR-431表达载体和SMAD4 3′UTR荧光素酶报告基因体系，论证不同时间点下对TGF-β通路的调控作用；（3）构建miR-431慢病毒表达载体，研究对不同时间点AECII增殖、转分化、凋亡及SPs合成与分泌的影响。因未见miR-431参与肺发育的报道，故可发现新的调控学说、并为某些肺发育疾病的机制研究提供共性理论基础。

新生儿肺发育机制至今未能完全明了。前期研究：miR-431为肺发育中筛选出的一条连续差异miRNA，同时高表达于RDS患儿血清及肺组织，抑制AECII增殖、致SP-B 表达下调；经预实验及信息学分析SMAD4为其重要靶基因、SMAD4参与的TGF-β通路为肺发育重要通路，提示miR-431可借由TGF-β/SMAD4通路参与肺发育及相关疾病的调控。本研究动态观察了不同时间点大鼠肺发育形态和miR-431、TGF-β通路连续表达变化，发现随着胎龄的增加，miR-431的表达量逐渐降低，而SMAD4、SP-A、SP-B、SP-C、SP-D mRNA及蛋白水平相应逐渐升高；构建荧光素酶载体证明了SMAD4为miR-431靶基因；通过在II型肺泡上皮细胞中干预miR-431了解了其表达情况及对II型上皮细胞增殖、凋亡的影响，发现过表达miR-431可加速A549细胞增殖，同时阻止其凋亡；抑制 miR-431表达则发现A549细胞增殖受阻，但凋亡方面与空白对照组相比无明显差异；借由挽救实验系统掌握了miR-431对下游TGF-β通路的具体调控作用，发现联合共转染miR-431 mimic及LV-SMAD4-至A549细胞后导致BMP4表达增加，而SPs mRNA水平减低。与之相对，转染miR-431 inhibitor及LV-SMAD4-后BMP4表达降低，而SPs mRNA水平增高。过表达miR-431及LV-SMAD4+联合共转染A549细胞后显示SPs水平减低，BMP4 mRNA及蛋白水平表达增加，与之相较，抑制miR-431及LV-SMAD4+联合共转染后SPs表达增加，BMP4 mRNA及蛋白水平表达减低。本课题为新生儿肺发育提供了重要理论基础，也为肺发育相关疾病的病理生理机制研究提供了新的思路。