miRNA Biogenesis Enzyme Drosha Is Required for Vascular Smooth Muscle Cell Survival

miRNA biogenesis enzyme Drosha cleaves double-stranded primary miRNA by interacting with double-stranded RNA binding protein DGCR8 and processes primary miRNA into precursor miRNA to participate in the miRNA biogenesis pathway. The role of Drosha in vascular smooth muscle cells (VSMCs) has not been well addressed. We generated Drosha conditional knockout (cKO) mice by crossing VSMC-specific Cre mice, SM22-Cre, with Drosha loxp/loxp mice. Disruption of Drosha in VSMCs resulted in embryonic lethality at E14.5 with severe liver hemorrhage in mutant embryos. No obvious developmental delay was observed in Drosha cKO embryos. The vascular structure was absent in the yolk sac of Drosha homozygotes at E14.5. Loss of Drosha reduced VSMC proliferation in vitro and in vivo. The VSMC differentiation marker genes, including αSMA, SM22, and CNN1, and endothelial cell marker CD31 were significantly downregulated in Drosha cKO mice compared to controls. ERK1/2 mitogen-activated protein kinase and the phosphatidylinositol 3-kinase/AKT were attenuated in VSMCs in vitro and in vivo. Disruption of Drosha in VSMCs of mice leads to the dysregulation of miRNA expression. Using bioinformatics approach, the interactions between dysregulated miRNAs and their target genes were analyzed. Our data demonstrated that Drosha is required for VSMC survival by targeting multiple signaling pathways.微小RNA（miRNA）生成酶Drosha可通过与双链RNA结合蛋白DGCR8相互作用，切割双链初级微小RNA（primary miRNA），并将其加工为前体微小RNA（precursor miRNA），进而参与miRNA生成通路。目前Drosha在血管平滑肌细胞（vascular smooth muscle cells, VSMCs）中的功能尚未得到充分阐明。本研究通过将血管平滑肌细胞特异性Cre工具小鼠SM22-Cre与Drosha flox/flox（loxp/loxp）小鼠杂交，成功构建了Drosha条件性敲除（conditional knockout, cKO）小鼠。在血管平滑肌细胞中敲除Drosha会导致胚胎于胚胎发育第14.5天（E14.5）死亡，突变胚胎出现严重的肝脏出血症状；且Drosha条件性敲除胚胎未出现明显的发育迟缓现象。在胚胎发育第14.5天，Drosha纯合子小鼠的卵黄囊中未检测到血管结构。Drosha缺失可在体外及体内抑制血管平滑肌细胞的增殖能力。与对照组相比，Drosha条件性敲除小鼠的血管平滑肌细胞分化标志物基因（包括αSMA、SM22及CNN1）以及内皮细胞标志物CD31的表达均显著下调。ERK1/2丝裂原活化蛋白激酶（mitogen-activated protein kinase, MAPK）与磷脂酰肌醇3-激酶/AKT（PI3K/AKT）信号通路在体外及体内的血管平滑肌细胞中均被抑制。小鼠血管平滑肌细胞中Drosha的缺失会导致miRNA表达失调。本研究通过生物信息学方法，对表达失调的miRNA与其靶基因之间的相互作用进行了分析。本研究数据表明，Drosha通过调控多条信号通路，对血管平滑肌细胞的存活至关重要。