ACTA VETERINARIA ET ZOOTECHNICA SINICA ›› 2018, Vol. 49 ›› Issue (11): 2371-2383.doi: 10.11843/j.issn.0366-6964.2018.11.008

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Study on the Regulation of Mouse miR-487b-3p in C2C12 Proliferation and Differentiation

LING Xiao-xiao1, TANG Peng1,2, JIA Cong-jun1, LIANG Chun-nian1, WU Xiao-yun1, CHU Min1, YAN Ping1*   

  1. 1. Key Laboratory of Yak Breeding Engineering of Gansu Province, Lanzhou Institute of Husbandry and Pharmaceutical Sciences of Chinese Academy of Agricultural Sciences, Lanzhou 730050, China;
    2. College of Animal Science and Technology, Gansu Agricultural University, Lanzhou 730050, China
  • Received:2018-04-02 Online:2018-11-23 Published:2019-01-23

Abstract:

This study aimed to explore the regulatory mechanism of miR-487b-3p on proliferation and differentiation of mouse C2C12 cells. In this study,mouse myoblast cell line(C2C12) was used as the research material,qRT-PCR was used to detect the expression of miR-487b-3p in various tissues and in C2C12 cells during proliferation and differentiation in mouse. After transfection of miR-487b-3p mimics and miR-487b-3p inhibitor,the transfection efficiency was detected by qRT-PCR,and the expression of proliferation marker gene PCNA and differentiation marker genes MYOD, MYOG and MYHC were detected by qRT-PCR and Western blotting. The bioinformatics softwares were used to predict target genes of miR-487b-3p,and the expression differences of PITX2 were detected by qRT-PCR in the proliferation and differentiation of mouse C2C12 cells,and the expression of PITX2 after over-expression(inhibition-expression) of miR-487b-3p was detected,and target relationship between miR-487b-3p and PITX2 was verified via the Dual-luciferase reporter assay. The results showed that the expression of miR-487b-3p was the highest in mouse skeletal muscle, and its expression level increased during the proliferation and differentiation of C2C12 cells. It was speculated that miR-487b-3p participated in the regulation of skeletal muscle development. Over-expression of miR-487b-3p significantly up-regulated the expression of miR-487b-3p(P<0.01), and significantly up-regulated the expression of PCNA at transcription and protein levels(P<0.01),and significantly down-regulated MYHC, MYOD and MYOG genes expression(P<0.05), simultaneously, significantly down-regulated the expression of MYHC(P<0.05),MYOD(P<0.01) and MYOG(P<0.05) proteins; After inhibiting the expression of miR-487b-3p, the expression of miR-487b-3p was significantly down-regulated(P<0.01),and the expression of PCNA gene was significantly down-regulated(P<0.05),and the expression of PCNA protein was significantly down-regulated(P<0.01),MYHC,MYOD and MYOG proteins expression were significantly up-regulated(P<0.05). The expression levels of MYHC,MYOD and MYOG genes increased,but the change was not significant(P>0.05). In the process of proliferation and differentiation of C2C12 cells, the expression trends of miR-487b-3p and PITX2 was reversed;Over-expression of miR-487b-3p could significantly down-regulate PITX2 mRNA expression(P<0.01),on the contrary,inhibition of miR-487b-3p could significantly up-regulate PITX2 mRNA expression(P<0.01). Dual-luciferase reporter assay verified that PITX2 was a direct target gene of miR-487b-3p. In summary,miR-487b-3p promotes the proliferation and inhibits the differentiation of C2C12 cells by targeting PITX2 in mouse.

Key words: mouse, miR-487b-3p, proliferation, differentiation, PITX2

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