circ-ZNF609 regulates G1-S progression in Rhabdomyosarcoma. circ-ZNF609 regulates G1-S progression in Rhabdomyosarcoma

Circular RNAs (circRNAs) represent a class of covalently closed RNAs, derived from a non-canonical splicing event, ubiquitously expressed among Eukaryotes and conserved among different species. We identified a circRNA (circ-ZNF609) involved in the regulation of human primary myoblast proliferation. Upon its depletion, the percentage of proliferating myoblasts was highly reduced. To deepen our knowledge about circ-ZNF609 role in cell cycle regulation, we studied its expression and function in Rhabdomyosarcoma (RMS), a pediatric skeletal muscle malignancy. We found that circ-ZNF609 is up-regulated in biopsies from both the two major RMS subtypes, the alveolar (ARMS) and the embryonal (ERMS), and we discovered that its knock-down blocks proliferation of an ERMS-derived cell line, while it has no effect on ARMS-derived cells. To understand the mechanism through which circ-ZNF609 affects cell proliferation we compared the different effects of circ-ZNF609 depletion in ERMS and ARMS and we identified genes and pathways on which the circRNA acts. Overall design: Total RNA from wild-type human primary myoblasts treated either with a control siRNA (si-SCR) or with a siRNA specific for circ-ZNF609 (si-Circ) was analyzed by RNA-Seq. The experiment was performed in duplicate (4 samples in total). Total RNA from RD cells (Embryonal Rhabdomyosarcoma) and RH4 cells (Alveolar Rhabdomyosarcoma) treated either with a control siRNA (si-SCR) or with a siRNA specific for circ-ZNF609 (si-Circ) was analyzed by RNA-Seq. The experiment was repeated in duplicate (8 samples in total).

环状RNA（circular RNAs, circRNAs）是一类经由非经典剪接事件产生的共价闭合RNA，广泛表达于真核生物中且在不同物种间保守。本研究团队鉴定到一种环状RNA circ-ZNF609，其参与调控人原代成肌细胞的增殖；当该环状RNA被敲低后，增殖期成肌细胞的比例显著降低。为深入探究circ-ZNF609在细胞周期调控中的作用，我们在横纹肌肉瘤（Rhabdomyosarcoma, RMS）——一种儿童骨骼肌恶性肿瘤——中研究了其表达与功能。我们发现，circ-ZNF609在两种主要横纹肌肉瘤亚型即腺泡状横纹肌肉瘤（alveolar RMS, ARMS）与胚胎性横纹肌肉瘤（embryonal RMS, ERMS）的活检组织中均呈上调表达；同时我们发现，敲低circ-ZNF609可阻断ERMS来源细胞系的增殖，但对ARMS来源细胞无明显作用。为阐明circ-ZNF609影响细胞增殖的分子机制，我们比较了其在ERMS与ARMS中敲低后的不同效应，并鉴定出该环状RNA作用的靶基因与通路。整体实验设计：分别对经对照小干扰RNA（si-SCR）或靶向circ-ZNF609的特异性小干扰RNA（si-Circ）处理的野生型人原代成肌细胞的总RNA进行RNA测序，实验设置生物学重复两次，共包含4个样本。另外，对RD细胞（胚胎性横纹肌肉瘤细胞系）与RH4细胞（腺泡状横纹肌肉瘤细胞系）分别采用si-SCR或si-Circ处理后提取总RNA，同样进行RNA测序，该实验重复两次，共包含8个样本。