Joint Laboratory of Reproductive Medicine, SCU-CUHK, Key Laboratory of Obstetric, Gynaecologic and Paediatric Diseases and Birth Defects of Ministry of Education, West China Second University Hospital, Sichuan University, Chengdu 610041, P. R. China.
Department of Obstetrics and Gynaecology, West China Second University Hospital, Sichuan University, Chengdu 610041, P. R. China.
Reprod Fertil Dev. 2020 Feb;32(4):355-362. doi: 10.1071/RD18474.
Circular RNAs belong to a new category of non-coding RNAs, characterised by a circular structure, conservation, stability and high expression in eukaryotes. They often show tissue- or cell-specific expression. Here, we identified a testis-enriched circular RNA (circRNA), circular Bbs9 (circ-Bbs9) that is highly expressed in mouse testis. An RNase R treatment experiment confirmed that circ-Bbs9 is indeed a circRNA. In situ hybridisation experiments showed that circ-Bbs9 is expressed in Leydig cells along seminiferous tubules and in the cytoplasm of the TM3 Leydig cell line. Knocking down the circ-Bbs9 in TM3 cells by lentivirus vectors arrested cell proliferation, whereas overexpression of circ-Bbs9 induced cell proliferation significantly. Knocking down circ-Bbs9 inhibited the protein level of cyclin D2 (Ccnd2) and RNA immunoprecipitation results showed that circ-Bbs9 interacts with Ccnd2. Our results show that use of the Hedgehog pathway Smoothened Agonist (SAG) HCl and antagonists cyclopamine and gant6 affects the expression levels of Glioma-Associated Oncogene Homolog 1 (Gli1), Ccnd2 and other genes in this pathway. Our research reveals that a Leydig cell-specific circRNA, circ-Bbs9, plays a critical role in Leydig cell proliferation through regulating the levels of cell cycle-related Ccnd2. Thus, our results emphasise the important role of circRNA in the male reproductive system.
环状 RNA 属于一种新的非编码 RNA 类别，其特征为环状结构、保守性、稳定性以及在真核生物中高表达。它们通常表现出组织或细胞特异性表达。在这里，我们鉴定了一种睾丸丰富的环状 RNA（circRNA），即环状 Bbs9（circ-Bbs9），它在小鼠睾丸中高度表达。RNase R 处理实验证实 circ-Bbs9 确实是一种 circRNA。原位杂交实验显示，circ-Bbs9 沿生精小管在 Leydig 细胞中以及在 TM3 Leydig 细胞系的细胞质中表达。通过慢病毒载体敲低 TM3 细胞中的 circ-Bbs9 会导致细胞增殖停滞，而过表达 circ-Bbs9 则显著诱导细胞增殖。敲低 circ-Bbs9 抑制了细胞周期蛋白 D2（Ccnd2）的蛋白水平，而 RNA 免疫沉淀结果表明 circ-Bbs9 与 Ccnd2 相互作用。我们的结果表明，使用 Hedgehog 通路 Smoothened 激动剂（SAG）HCl 以及拮抗剂 cyclopamine 和 gant6 会影响该通路中 Glioma-Associated Oncogene Homolog 1（Gli1）、Ccnd2 和其他基因的表达水平。我们的研究揭示了一种睾丸特异性环状 RNA，circ-Bbs9，通过调节细胞周期相关 Ccnd2 的水平，在 Leydig 细胞增殖中发挥关键作用。因此，我们的结果强调了 circRNA 在男性生殖系统中的重要作用。
