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

Citation: Fan P, Chen Z, Tian P, Liu W, Jiao Y, et al. ( miRNA Biogenesis Enzyme Drosha Is Required for Vascular Smooth Muscle Cell Survival Pei Fan 0 Zixuan Chen 0 Peng Tian 0 Wen Liu 0 Yan Jiao 0 Yi Xue 0 Anindya Bhattacharya 0 Jianmin Wu 0 Meifen Lu 0 Yuqi Guo 0 Yan Cui 0 Weikuan Gu 0 Weiwang Gu 0 Junming Yue 0 Maurizio Pesce, Centro Cardiologico Monzino, Italy 0 1 Department of Pathology, University of Tennessee Health Science Center, Memphis, Tennessee, United States of America, 2 Center for Cancer Research, University of Tennessee Health Science Center, Memphis, Tennessee, United States of America, 3 Department of Orthopaedic Surgery-Campbell Clinic, University of Tennessee Health Science Center, Memphis, Tennessee, United States of America, 4 Department of Neurology, University of Tennessee Health Science Center, Memphis, Tennessee, United States of America, 5 Southern Medical University , Guangzhou, Guangdong , P. R. China , 6 The Third Affiliated Hospital of Zhengzhou University , Zhengzhou, Henan , P. R. China , 7 Department of Microbiology, Immunology and Biochemistry, University of Tennessee Health Science Center, Memphis, Tennessee, United States of America, 8 Veterinary Research Institute , Nanning, Guangxi , P. R. China 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 aSMA, 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. - Funding: This work was supported by awards HL095957 and HD061420 to J. Yue from NHLBI, the Eunice Kennedy Shriver National Institute of Child Health & Human Development, and by UTHSC startup funding as well as by awards S2012ZR0233,1021010200004, 2010A011200003 and 2012B040304010 to W. Gu from Chinese government. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: Junming Yue is an academic editor of PLOS ONE. This does not alter the authors adherence to all the PLOS ONE policies on sharing data and materials. Drosha is an RNAase III enzyme that interacts with the RNA binding protein DGCR8 and forms a microcomplex in the nucleus to process primary miRNAs (pri-miRNAs) into precursor miRNAs (pre-miRNAs), which are subsequently cleaved by Dicer in the cytoplasm to produce mature miRNA. Accumulating experimental evidence suggests that miRNAs control gene expression through RNA-induced silencing complex (RISC) at the posttranscriptional level and regulates cell apoptosis, proliferation, and differentiation [1,2,3,4]. The miRNA biogenesis pathway is tightly controlled through different molecular mechanisms including methylation, RNA editing, and a self-regulatory feedback loop between Drosha and DGCR8 in the physiological condition [5]. Disruption of the miRNA biogenesis pathway causes aberrant expression profiles of miRNA genes, thus leading to various human diseases and developmental retardation. DGCR8 specifically processes miRNA, whereas Dicer functions in other small RNAs such as siRNA or shRNA. in addition to miRNAs. Recent studies indicated that some splicing-independent mirtron-like miRNA (simtrons) maturation does not require the canonical miRNA biogenesis pathway, which skips the processing of DGCR8, exportin-5, Dicer, and Ago2, but is Drosha-dependent [6,7,8]. In our previous studies, we showed that disruption of DGCR8 in VSMCs results in embryonic death 23 days earlier than that of Dicer, although both DGCR8 and Dicer cKO embryos share some phenotypic similarity [9,10]. Our studies demonstrated that DGCR8 plays a more important role than does Dicer in maintaining the VSMC functions by participating in the miRNA process in the upstream biogenesis pathway. Drosha and Dicer are two RNAase III enzymes and share the structural and functional similarity that both cleave the double-stranded RNA and have two RNAase III domains, a/b and an RNA binding domain. However, DGCR8 is an RNA binding protein whose structure differs from Dicer and Drosha, which do not have RNAase III domains but have two RNA binding domains. Drosha, DGCR8, and Dicer are key components of the miRNA biogenesis pathway and contribute to miRNA maturation by participating in this pathway. In our previous studies [9,10], we have shown that DGCR8 and Dicer are required for vascular development. However, the roles of Drosha in VSMCs are largely unknown. To investigate the role of Drosha in VSMC function, we generated conditional Drosha knockout mice (cKO) by crossing Drosha loxp/loxp with VSMCspecific SM22-cre mice. Disruption of Drosha in VSMCs of mice results in embryonic mortality. In addition, we also generated KO VSMCs by using adeno-cre virus and knockdown (KD) VSMCs using retroviral shRNA vector. By characterizing Drosha cKO mice, KO, and KD VSMCs, we found that loss of Drosha in VSMCs reduces VSMC proliferation and differentiation by compromising miRNA biogenesis pathways and attenuating cellular survival pathways ERK1/2 and AKT. Loss of Drosha in VSMCs Results in Embryonic Lethality To determine the role of Drosha in VSMCs, we generated Drosha cKO mice by crossing Droshaloxp/loxp mice with VSMCspecific SM22-Cre transgenic mice. Exon 9 of Drosha was floxed and deleted through Cre-LoxP recombination, thus leading to disrupting Drosha expression in VSMCs of mice (Figure S1A). Of 120 live pups genotyped, no homozygote Drosha mice were found, suggesting that loss of Drosha in VSMCs of mice results in embryonic lethality. The Drosha heterozygotes were viable and did not display any developmental abnormalities. Drosha homozygotes died at embryonic day E14.5 without obvious developmental delay (Figure 1A). The vasculature was severely impaired in the yolk sacs of Drosha cKO mice after E14.5; no blood vessels (...truncated)