The THO Complex-Dependent Posttranscriptional Control Contributes to Vascular Smooth Muscle Cell Fate Decision. The THO Complex-Dependent Posttranscriptional Control Contributes to Vascular Smooth Muscle Cell Fate Decision

We applied RNA-Seq to analyze the effects of silencing of Thoc2 or Thoc5, two components of the THO complex, in cultured VSMC. The result revealed that Thoc5 silencing closely resembled the gene expression changes induced upon PDGF-BB/PDGF-DD treatments in cultured VSMCs. Mechanistically, our RIP-Seq data revealed both Thoc2 and Thoc5 preferentially interacted with VSMC marker gene mRNAs and mediated their expression. Interestingly, mRNAs that lost Thoc2 or Thoc5 binding during VSMC dedifferentiation were enriched for genes important for VSMC identity. In addition, silencing of Thoc2 or Thoc5 led to dedifferentiation of VSMCs in vitro, characterized by decreased VSMC marker gene expression and increased migration and proliferation. Furthermore, we performed immuno-histochemical staining against Thoc2 and Thoc5, and found a dramatic reduction in their expression in human arteries undergoing carotid endarterectomy (CEA) compared to normal internal mammary arteries (IMA). Notably, Thoc5 overexpression in injured rat carotid arteries significantly repressed loss of VSMC marker gene expression and neointima formation. Together, our data introduce dynamic binding of THO to VSMC marker gene mRNAs as a novel mechanism contributing to VSMC fate decisions, and imply Thoc5 as a potential intervention node for vascular diseases. Overall design: RNA-Seq of Thoc2 or Thoc5 silencing in cultured mVSMC; ChIP-seq of Thoc5 in wild-type cultured mVSMC; RIP-Seq of Thoc2 or Thoc5 in cultured mVSMC.

本研究采用RNA测序（RNA-Seq）分析了敲低THO复合物（THO complex）的两个组分Thoc2与Thoc5对体外培养的血管平滑肌细胞（VSMC）的影响。实验结果显示，Thoc5沉默所诱导的基因表达变化，与血小板衍生生长因子-BB（PDGF-BB）/血小板衍生生长因子-DD（PDGF-DD）处理体外培养VSMC后的基因表达变化高度相似。从机制层面而言，本研究的RNA免疫沉淀测序（RIP-Seq）数据表明，Thoc2与Thoc5均优先结合VSMC标志物基因的mRNA，并调控其表达。有趣的是，在VSMC去分化过程中失去Thoc2或Thoc5结合的mRNA，显著富集于维持VSMC身份特征的关键基因中。此外，体外敲低Thoc2或Thoc5可诱导VSMC发生去分化，具体表现为VSMC标志物基因表达下调，同时细胞迁移与增殖能力增强。进一步地，本研究针对Thoc2与Thoc5开展了免疫组织化学染色，发现接受颈动脉内膜剥脱术（CEA）的患者动脉标本中，二者的表达水平较正常乳内动脉（IMA）出现显著降低。值得注意的是，在受损大鼠颈动脉中过表达Thoc5，可显著抑制VSMC标志物基因表达的丢失以及新内膜形成。综上，本研究揭示了THO复合物与VSMC标志物基因mRNA的动态结合是调控VSMC命运决定的全新机制，并提示Thoc5可作为血管疾病潜在的干预靶点。实验设计概述：体外培养小鼠血管平滑肌细胞（mVSMC）中Thoc2或Thoc5沉默后的RNA测序；野生型体外培养mVSMC中Thoc5的染色质免疫沉淀测序（ChIP-Seq）；体外培养mVSMC中Thoc2或Thoc5的RNA免疫沉淀测序（RIP-Seq）。