Testis-enriched circular RNA circ-Bbs9 plays an important role in Leydig cell proliferation by regulating a CyclinD2-dependent pathway
Minzhi Jia A * , Xiaoliang Li A * , Chuan Jiang A , Ke Wang A , Tao Zuo A , Guolin He B , Lang Qin B C C and Wenming Xu
A B C
A 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.
B Department of Obstetrics and Gynaecology, West China Second University Hospital, Sichuan University, Chengdu 610041, P. R. China.
C Reproductive Medical Center, Department of Obstetrics and Gynecology, West China Second University Hospital, Sichuan University, Chengdu 610041, China.
D Corresponding authors. Email: xuwenming@scu.edu.cn; cacier@163.com
Reproduction, Fertility and Development 32(4) 355-362 https://doi.org/10.1071/RD18474
Submitted: 21 November 2018 Accepted: 1 July 2019 Published: 11 November 2019
Abstract
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.
Additional keywords: cell cycle, cyclin D2, Leydig cell, proliferation.
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