目的：内脏平滑肌细胞（SMC）是胃肠道（GI）中调节胃肠运动的组成部分。SMC收缩受翻译后信号和分化状态的调节。SMC收缩受损与显著的发病率和死亡率相关，但是调节SMC特异性收缩基因表达的机制，包括长链非编码RNA（lncRNAs）的作用，在很大程度上仍未被探索。在这里，我们揭示了Carmn（心脏中胚层增强子相关非编码RNA）的关键作用，SMC特异性lncRNA，在调节内脏SMC表型和胃肠道收缩性方面。
方法：来自胚胎的GTEx和公开可用的单细胞RNA测序（scRNA-seq）数据集，询问成人和小鼠GI组织以鉴定SMC特异性lncRNAs。使用新型GFP敲入(KI)报告子/敲除(KO)小鼠研究了Carmn的功能作用。结肠肌层的大量RNA测序（RNA-seq）和单核RNA测序（snRNA-seq）用于研究潜在的机制。
结果：在CarmnGFPKI小鼠中的无偏倚的计算机分析和GFP表达模式揭示了Carmn在人和小鼠的GISMC中高度表达。由于胃肠道假性阻塞，在全球CarmnKO（gKO）和诱导型SMC特异性KO（iKO）小鼠中观察到过早的致死性，盲肠和结肠段有动力障碍的胃肠道严重扩张。组织学,胃肠道运输和肌肉肌电图分析显示严重扩张，与对照小鼠相比，CarmnKO的GI转运显着延迟和GI收缩性受损。GI肌层的BulkRNA-seq显示，Carmn的缺失促进SMC表型转换，细胞外基质基因的上调和SMC收缩基因的下调证明了这一点。包括Mylk,SMC收缩的关键调节器。snRNA-seq进一步揭示了SMCCarmnKO不仅通过减少收缩基因表达而损害了肌源性运动，而且通过破坏结肠肌层中的细胞-细胞连接而损害了神经源性运动。这些发现可能具有翻译意义，因为沉默人结肠SMC中的CARMN显着减弱了收缩基因表达，包括MYLK,和SMC收缩性降低。荧光素酶报告基因分析显示CARMN增强SMC收缩表型主调节因子的反式激活活性,myocardin,从而维持GISMC生肌程序。
结论：我们的数据表明，Carmn对于维持小鼠GISMC收缩功能是必不可少的，CARMN功能的丧失可能导致人类内脏肌病。据我们所知，这是第一项研究显示lncRNA在内脏SMC表型调节中的重要作用。
Visceral smooth muscle cells (SMCs) are an integral component of the gastrointestinal (GI) tract that regulate GI motility. SMC contraction is regulated by posttranslational signaling and the state of differentiation. Impaired SMC contraction is associated with significant morbidity and mortality, but the mechanisms regulating SMC-specific contractile gene expression, including the role of long noncoding RNAs (lncRNAs), remain largely unexplored. Herein, we reveal a critical role of Carmn (cardiac mesoderm enhancer-associated noncoding RNA), an SMC-specific lncRNA, in regulating visceral SMC phenotype and contractility of the GI tract.
Genotype-Tissue Expression and publicly available single-cell RNA sequencing (scRNA-seq) data sets from embryonic, adult human, and mouse GI tissues were interrogated to identify SMC-specific lncRNAs. The functional role of
Carmn was investigated using novel green fluorescent protein (GFP) knock-in (KI) reporter/knock-out (KO) mice. Bulk RNA-seq and single nucleus RNA sequencing (snRNA-seq) of colonic muscularis were used to investigate underlying mechanisms.
Unbiased in silico analyses and GFP expression patterns in
Carmn GFP KI mice revealed that
Carmn is highly expressed in GI SMCs in humans and mice. Premature lethality was observed in global Carmn KO and inducible SMC-specific KO mice due to GI pseudo-obstruction and severe distension of the GI tract, with dysmotility in cecum and colon segments. Histology, GI transit, and muscle myography analysis revealed severe dilation, significantly delayed GI transit, and impaired GI contractility in Carmn KO vs control mice. Bulk RNA-seq of GI muscularis revealed that loss of
Carmn promotes SMC phenotypic switching, as evidenced by up-regulation of extracellular matrix genes and down-regulation of SMC contractile genes, including Mylk, a key regulator of SMC contraction. snRNA-seq further revealed SMC Carmn KO not only compromised myogenic motility by reducing contractile gene expression but also impaired neurogenic motility by disrupting cell-cell connectivity in the colonic muscularis. These findings may have translational significance, because silencing CARMN in human colonic SMCs significantly attenuated contractile gene expression, including MYLK, and decreased SMC contractility. Luciferase reporter assays showed that CARMN enhances the transactivation activity of the master regulator of SMC contractile phenotype, myocardin, thereby maintaining the GI SMC myogenic program.
Our data suggest that
Carmn is indispensable for maintaining GI SMC contractile function in mice and that loss of function of
CARMN may contribute to human visceral myopathy. To our knowledge this is the first study showing an essential role of lncRNA in the regulation of visceral SMC phenotype.
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