Long noncoding RNA Hoxb3os is dysregulated in autosomal dominant polycystic kidney disease and regulates mTOR signaling. Long noncoding RNA Hoxb3os is dysregulated in autosomal dominant polycystic kidney disease and regulates mTOR signaling

Autosomal dominant polycystic kidney disease (ADPKD) is a debilitating disease that is characterized by the accumulation of numerous fluid-filled cysts in the kidney. ADPKD is primarily caused by mutations in two genes, PKD1 and PKD2. Long noncoding RNAs (lncRNA) – defined by a length >200 nucleotides and absence of a long open reading frame – have recently emerged as epigenetic regulators of development and disease; however, their involvement in PKD has not been explored previously. Here, we performed deep RNA sequencing to identify lncRNAs that are dysregulated in two orthologous mouse models of ADPKD (kidney-specific Pkd1 and Pkd2 mutant mice). We identified a kidney- specific, evolutionarily-conserved lncRNA called Hoxb3os that was downregulated in cystic kidneys from Pkd1 and Pkd2 mutant mice. The human ortholog HOXB3-AS1 was downregulated in cystic kidneys from ADPKD patients. Hoxb3os was highly expressed in renal tubules in adult wild- type mice, whereas its expression was lost in the cyst epithelium of mutant mice. To investigate the function of Hoxb3os, we utilized CRISR/Cas9 to knockout its expression in mIMCD3 cells. Deletion of Hoxb3os resulted in increased phosphorylation of mTOR and its downstream targets, including p70 S6 kinase, ribosomal protein S6, and the translation repressor 4E-BP1. Consistent with activation of mTORC1 signaling, Hoxb3os mutant cells displayed increased mitochondrial respiration. The Hoxb3os mutant phenotype was partially rescued upon re- expression of Hoxb3os in knockout cells. These findings identify Hoxb3os as a novel lncRNA that is downregulated in ADPKD and regulates mTOR signaling and mitochondrial respiration. Overall design: Expression profiling by sequencing in primary kidney and cultured cells.

常染色体显性遗传性多囊肾病（Autosomal dominant polycystic kidney disease, ADPKD）是一种致残性疾病，其特征为肾脏内大量充满液体的囊肿形成。ADPKD主要由PKD1和PKD2两个基因的突变所引发。长链非编码RNA（Long noncoding RNAs, lncRNA）——定义为长度超过200个核苷酸且不具备长开放阅读框的RNA分子——近年来已成为发育与疾病进程中的表观遗传调控因子；然而，此前尚未有研究探索其在多囊肾病中的作用。本研究通过深度RNA测序技术，在两种ADPKD同源小鼠模型（肾脏特异性Pkd1突变小鼠与Pkd2突变小鼠）中筛选表达失调的lncRNA。我们鉴定出一种肾脏特异性、进化保守的lncRNA，命名为Hoxb3os，该分子在Pkd1与Pkd2突变小鼠的囊性肾脏中表达下调。其人类同源基因HOXB3-AS1在ADPKD患者的囊性肾脏中同样呈现表达下调。Hoxb3os在成年野生型小鼠的肾小管中呈高表达，而在突变小鼠的囊肿上皮细胞中完全缺失表达。为探究Hoxb3os的生物学功能，我们利用CRISPR/Cas9技术在mIMCD3细胞中敲除其表达。敲除Hoxb3os会导致哺乳动物雷帕霉素靶蛋白（mammalian target of rapamycin, mTOR）及其下游靶点的磷酸化水平升高，这些靶点包括p70 S6激酶、核糖体蛋白S6以及翻译抑制因子4E-BP1。与mTOR复合物1（mTORC1）信号通路激活的表型一致，Hoxb3os敲除细胞表现出线粒体呼吸功能增强。在敲除细胞中重新表达Hoxb3os，可部分挽救该突变表型。本研究结果证实，Hoxb3os是一种在ADPKD中表达下调的新型lncRNA，可调控mTOR信号通路与线粒体呼吸功能。整体实验设计：通过测序技术对原代肾脏组织与培养细胞进行表达谱分析。