Effects Of Overexpression Of Six1 On The Expression Of Protein Metabolism-related Genes And Preliminary Study Of Its Regulatory Role In Duck Myoblasts

Six1 is an important members of Sine oculis homeobox protein family (SIX), plays a critical role in initiation of myogenesis, formation of myotome and differentiation of muscle fiber types. In addition, Six1 and its cofactor Eya2 directly up-regulate the expression of mTOR, thereby contributing to the enhancement of protein synthesis, causing physiological cardiac hypertrophy, revealed that Six1 also plays an important role in muscle protein synthesis, however, the specific regulatory mechanism in muscle protein metabolism has not been reported. In this study, we detected the effects of overexpression of Six1 via transfection of pEGFP-duSixl on the cellullar proliferative activity and protein metablism; moreover, in order to further investigate whether Six1 would play an regulatory role on PI3K and mTOR, we firstly treated the duck myoblasts in vitro using LY294002 and Rapamycin, a quite specific inhibitor of PI3K and mTOR, respectively, and subsequently transfected myoblasts with pEGFP-duSix1, to explore the possible regulatory mechanism of Six1 in the process of cellular protein metabolism. The main results are as follows:1. mRNA expression level of Sixl was significantly increased in duck myoblasts which were transfected with eukaryotic expression vector pEGFP-duSix1 than in control groups (P<0.01), meanwhile, the cell proliferative activity was significantly increased (P<0.05), while mRNA expression levels of mTOR and S6K1 were also significantly up-regulated after transfection (P<0.05); Additionally, the results of Western-blot showed that the protein expression of S6K1 was increased after transfection of pEGFP-duSixl, suggesting that overexpression of Six1 plays an important role in promoting protein synthesis and regulating of PI3K/Akt/mTOR signaling pathway in duck myoblasts.2. Our results revealed that mRNA expression levels of FOX01, MAFbx, MuRFl which were maker genes relevant to protein degradation were increased in Sixl-transfected groups than in control groups, but no significantly (P>0.05)5 Western-blot results also showed that protein expression of p-FOXO1 was increased, which speculate that overexpression of Six1 gene may also influence metabolic of cellular protein degradation pathways, but need for further studies.3. LY294002 and Rapamycin, a quite specific inhibitor of P13K and mTOR, were added into the medium of duck myoblasts cltured in vitro, and we found that the cell proliferative activity were significantly decreased (P<0.05), and with the increasing of treatment time and inhibitor concentration, the inhibitory effects were more obvious; thereinto, in the myoblasts which after LY294002 treated the mRNA expression levels of PI3K, Akt, mTOR were significantly decreased (P<0.05), whereas in Rapamycin-treated groups, only the expression of mTOR was significantly decreased (P<0.05), while the expression of PI3K, Akt were not significantly decreased (P>0.05). Western-blot results also showed that protein expression of S6K1 decreased, indicating that these two inhibitors could significantly inhibited protein synthesis in myoblasts; at the same time we found that the mRNA expression levels of FOXO1, MAFbx, MuRFl involved in protein degradation were not changed significantly.4. Duck myoblasts were transfected by pEGFP-duSix1 which treated with LY294002 and Rapamycin before, and showed that overexpression of Six1 can significantly recover the proliferative activity of myoblasts (P<0.05).Moreover, we found that overexpression of Six1 could rescue the inhibitory effects of cellular protein synthesis which caused by LY294002 and Rapamycin, showing a significantly increase in mRNA expression of mTOR, S6K1 (P<0.05), while the protein expression of S6K1 also showed an upward trend. However, the mRNA expression levels of FOXO1, MAFbx and MuRFl showed different trends in these two experiments.5. Duck myoblasts were treated with LY294002 and Rapamycin firstly, and then overexpressing of Six1 gene can significantly up-regulated the mRNA expression levels of mTOR and its downstream S6K1 (P<0.05), whereas the mRNA expression levels of PI3K and Akt in myoblasts which were treated with LY294002 before were not increased but significantly reduced (P<0.05), suggesting that Six] directly promote the expression of mTOR to enhance protein synthesis, but has no obvious regulatory role on PI3K.