干扰Mtmr3基因对C2C12细胞增殖与分化的影响

doi:10.11843/j.issn.0366-6964.2023.04.012

Abstract

Abstract: This study aimed to investigate the regulation and mechanism of Mtmr3 (myotubularin related protein 3) on proliferation and differentiation of C2C12 cells. In this study, mouse myoblast cell line (C2C12) was used as experimental material. qRT-PCR and immunofluorescence staining were used to detect the mRNA expression level of Mtmr3 gene in myoblast growth and differentiation stages, and the distribution morphology on day 6 of differentiation. The small interfering RNA (siRNA) of Mtmr3 was synthesized and divided into siRNA NC control group and Mtmr3 siRNA treatment group (n=3). EdU, CCK-8, qRT-PCR and Western blot were used to detect the influence of Mtmr3 siRNA on myoblast proliferation and differentiation, and the relevant mechanisms of regulating myoblast proliferation and differentiation were studied through signal pathways. The results showed that the mRNA expression level of Mtmr3 decreased in the growth stage, but increased gradually in the differentiation stage, and the immunofluorescence staining of Mtmr3 showed a myotube shape. After interfering with Mtmr3, mRNA and protein expression levels of Mtmr3 gene were significantly decreased (P<0.01). In proliferation test, the percentage of EdU positive cells in total cells and cell viability were significantly increased after transfection of Mtmr3 siRNA (P<0.01). After the interference of Mtmr3 expression, the mRNA and protein expression levels of Pcna and Cdk4 were significantly increased (P<0.01), and the mRNA expression level of Ccnd gene was significantly increased (P<0.01). In the differentiation test, the expression level of differentiation marker gene Myhc protein was significantly inhibited after transfection of Mtmr3 siRNA (P<0.01), and the mRNA expression levels of Mtmr3, Myog and Myhc genes were significantly inhibited at the 4th and 6th day of cell differentiation (P<0.01). After interfering Mtmr3 expression in proliferative stage and differentiation stage, phosphorylated protein expression levels of Mtor and P70s6k were significantly increased and significantly decreased, respectively (P<0.01).The results of CCK-8 showed that Mtmr3 siRNA could counteract the inhibitory effect of Rapamycin (Rapa), a specific inhibitor of Mtor pathway, and promote myoblast proliferation compared with the control group (P<0.01); The mRNA expression levels of Mtor, P70s6k, Myog and Myhc genes were significantly decreased in myoblasts treated with Rapa during cell differentiation (P<0.01). In summary, the expression level of Mtmr3 in myoblast growth stage showed a downward trend, while that in differentiation stage showed a gradual upward trend, and the immunofluorescence staining of Mtmr3 showed a myotube shape. Interfering with Mtmr3 expression promotes myoblast proliferation by activating the Mtor pathway, and inhibits myoblast differentiation by inactivating the Mtor pathway.

Key words: myoblast, Mtmr3, proliferation, differentiation, Mtor

CLC Number:

S813.1

WANG Kaiming, YU Zonggang, XU Xueli, AI Nini, LI Xintong, HE Jun, TAO Deng, ZHANG Shuo, MA Haiming, ZHANG Yuebo. Effect of Interfering Mtmr3 on C2C12 Cells Proliferation and Differentiation[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(4): 1478-1489.

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Effect of Interfering Mtmr3 on C2C12 Cells Proliferation and Differentiation

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