MiR-2425-5p targets RAD9A and MYOG to regulate the proliferation and differentiation of bovine skeletal muscle-derived satellite cells.
Our group previously identified miR-2425-5p, a unique bovine miRNA; however, its biological function and regulation in muscle-derived satellite cells (MDSCs) remain unclear. Herein, stem-loop RT-PCR results showed that miR-2425-5p increased during MDSCs proliferation, but decreased during differentiation. Cell proliferation was examined using EdU assays, cyclin B1 (CCNB1) and proliferating cell nuclear antigen (PCNA) western blot (WB) and flow cytometry analysis. These results showed that miR-2425-5p mimics (miR-2425-M) enhanced MDSCs proliferation, whereas, miR-2425-5p inhibitor (miR-2425-I) had opposite effect. Conversely, cell differentiation studies by desmin (DES) immunofluorescence, myotubes formation, and myosin heavy chain 3 (MYH3) WB analyses revealed that miR-2425-M and miR-2425-I blocked and promoted MDSCs differentiation, respectively. Moreover, luciferase reporter, RT-PCR, and WB assays showed that miR-2425-5p directly targeted the 3'-UTR of RAD9 homolog A (RAD9A) and myogenin (MYOG) to regulate their expression. Rescue experiment showed RAD9A inhibited the proliferation of MDSCs through miR-2425-5p. In addition, we found that miR-2425-5p expression was regulated by its host gene NCK associated protein 5-like (NCKAP5L) rather than being transcribed independently as a separate small RNA. Collectively, these data indicate that miR-2425-5p is a novel regulator of bovine MDSCs proliferation and differentiation and provides further insight into the biological functions of miRNA in this species.
The Laboratory of Cell and Developmental Biology, Northeast Agricultural University, Harbin, Heilongjiang, 150030, China.
Hui Li
HL
Tong
The Laboratory of Cell and Developmental Biology, Northeast Agricultural University, Harbin, Heilongjiang, 150030, China.
Run Ying
RY
Jiang
College of Life Sciences and Agriculture & Forestry, Qiqihar University, Qiqihar, Heilongjiang, 161006, China.
Wei Wei
WW
Zhang
The Laboratory of Cell and Developmental Biology, Northeast Agricultural University, Harbin, Heilongjiang, 150030, China. yanyunqin@sohu.com.
Yun Qin
YQ
Yan
D000818:Animals; D002417:Cattle; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002454:Cell Differentiation; D049109:Cell Proliferation; D005786:Gene Expression Regulation; D035683:MicroRNAs / Q000378:metabolism*; D024510:Muscle Development; D018008:Myogenin / Q000378:metabolism*; D032496:Satellite Cells, Skeletal Muscle / Q000378:metabolism*
D018797:Cell Cycle Proteins; D035683:MicroRNAs; D018008:Myogenin; C069930:rad9 protein