The Circular RNA circHUWE1 Sponges the miR-29b-AKT3 Axis to Regulate Myoblast Development

Binglin Yue; Jian Wang; Wenxiu Ru; Jiyao Wu; Xiukai Cao; Haiyan Yang; Yongzheng Huang; Xianyong Lan; Chuzhao Lei; Bizhi Huang; Hong Chen

doi:10.1016/j.omtn.2019.12.039

The Circular RNA circHUWE1 Sponges the miR-29b-AKT3 Axis to Regulate Myoblast Development

Mol Ther Nucleic Acids. 2020 Mar 6:19:1086-1097. doi: 10.1016/j.omtn.2019.12.039. Epub 2020 Jan 14.

Authors

Affiliations

¹ Key laboratory of Animal Genetics, Breeding and Reproduction of Shaanxi Province, College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi 712100, China.
² Yunnan Academy of Grassland and Animal Science, Kunming, Yunnan 650212, China.
³ Key laboratory of Animal Genetics, Breeding and Reproduction of Shaanxi Province, College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi 712100, China. Electronic address: chenhong1212@263.net.

Abstract

Myogenesis is controlled by a well-established transcriptional hierarchy that coordinates the activities of a set of muscle genes. Recently, roles in myogenesis have been described for non-coding RNAs, including a role of circular RNA (circRNA) to regulate muscle gene expression. However, the functions of circRNA and the underlying mechanism by which circRNAs affect myogenesis remain poorly understood. In this study, we analyzed circRNA high-throughput sequencing results of bovine skeletal muscle samples and constructed a circRNA-miRNA-mRNA network according to the competitive endogenous RNA (ceRNA) theory. The putative circHUWE1-miR-29b-AKT3 network was analyzed and its involvement in myogenesis was confirmed through a series of assays. To assess the potential function of this regulation, bovine myoblasts were infected with overexpression plasmids and small interfering RNAs (siRNAs) that target circHUWE1. Next, cell proliferation, apoptosis, and differentiation were analyzed using Cell Counting Kit-8 (CCK-8), 5-ethynyl-2'-deoxyuridine (EdU), flow cytometry, western blotting, and qRT-PCR assays. The results suggest that circHUWE1 facilitates bovine myoblast proliferation and inhibits cell apoptosis and differentiation. Next, bioinformatics, dual-luciferase reporter assay, and AGO2 RNA immunoprecipitation (RIP) approaches were used to verify the interaction between circHUWE1, miR-29b, and AKT3. Subsequently, we identified that circHUWE1 could directly interfere with the ability of miR-29b to relieve AKT3 suppression, which ultimately activates the AKT signaling pathway. These findings suggested a new regulatory pathway for bovine skeletal muscle development, and they also expand our understanding of circRNA functions in mammals.

Keywords: ceRNA; circHUWE1-miR-29b-AKT3; skeletal muscle.