Effect Of Leucine And MiR-27a On Porcine Myoblast Differentiation And Myofiber Type Transformation And Its Mechanism

Leucine,one of the branched-chain amino acid,regulates the turnover of skeletal muscle proteins and plays an important role in myogenesis.Mirco RNA-27a(miR-27a)plays a vital effect during the devevopment of skeletal muscle.It has been reported that nutrients could modulate biological function through regulating the expression of endogenous micro RNA.However,the effect of leucine and miR-27 a on porcine myoblast differentiation and myofiber type transformation are still unknown.Moreover,whether leucine regulates porcine myofiber type transformation through miR-27 a is also unclear.Therefore,the object of this study is to investigate the effect of leucine and miR-27 a on porcine myoblast differentiation and myofiber type transformation and explore its mechanism.The research contains five parts of experimets are as follows: Exp.1 Effect of leucine on porcine myoblast differentiation and its mechanismIn this study,in order to explore the role of leucine on porcine myoblast differentiation,different concentration of leucine was added to myoblasts for 3 days when cells were induced to differentiate.In addition,leucine and wortmannin,the specific repressor of Akt,were added to differentiation medium simultaneously to explore whether leucine regulates porcine myoblast differentiation through Akt/FoxO1 signaling pathway.The results showed that leucine increased the number of myosin heavy chain(MHC)-positive cells and CK activity.Myogenin and Myo D m RNA and protein expression levels were also increased.Leucine increased the levels of P-Akt/Akt and P-FoxO1/FoxO1,as well as decreased the protein level of FoxO1.The expression of miR-27 a was increased after leucine treatment.However,wortmannin attenuated the positive role of leucine on porcine myoblast differentiation.Our results suggested that leucine promotes porcine myoblast differentiation through Akt/FoxO1 signaling pathway.Exp.2 Effect of leucine on porcine myofiber type transformationand its mechanismTo study the effect of leucine on porcine myofiber type transformation,after 3 days of differentiation,porcine myotubes cultured in differentiation medium were treated with different concentration of leucine for 4 days.In addition,leucine and wortmannin were added to differentiation medium simultaneously to explore whether leucine regulates porcine myofiber type transformation through Akt/FoxO1 signaling pathway.The results showed that leucine increased MyHCI,MyHCIIa,MyHCIIx m RNA levels and slow MyHCprotein level,and decreased fast MyHCprotein level.Leucine increased the levels of P-Akt/Akt and P-FoxO1/FoxO1,as well as decreased the levels of FoxO1 and miR-27 a.However,blocking Akt/FoxO1 signal pathway by wortmannin attenuated the role of leucine on porcine myofiber type transformation.These results indicated that leucine promotes porcine myofiber type transformation form fast-twich to slow-tiwch through Akt/FoxO1 signaling pathway.Exp.3 Effect of miR-27 a on porcine myobalst differentiation and its mechanismTo investigate the effect of miR-27 a on differentiation of porcine myobalst,liposome transfection method was used to promote or repress the expression of miR-27 a.Furthermore,wortmannin and miR-27 a inhibitor were used to explore whether miR-27 a regulates porcine myobalst differentiation via Akt/FoxO1 signaling pathway.We found that overexpression of miR-27 a suppressed the myotube formation,decreased myogenin and Myo D m RNA and protein expression levels,as well as increased myostatin m RNA expression,whereas inhibition of miR-27 a had an opposite effect.In addition,overexpression of miR-27 a decreased the levels of P-Akt/Akt and P-FoxO1/FoxO1,increased the protein level of FoxO1,however,inhibition of miR-27 a had an opposite effect.However,wortmannin attenuated the role of miR-27 a on porcine myoblast differentiation.These results demonstrated that miR-27 a inhibits porcine myoblast differentiation via Akt/FoxO1 signaling pathway.Exp.4 Effect of miR-27 a on porcine myofiber type transformation and its mechanismTo investigate the effect of miR-27 a on porcine myofiber type transformation,after 3 days of differentiation,miR-27 a mimics and inhibitor were transfected into cells for 3 days.Furthermore,wortmannin and miR-27 a inhibitor were used to explore whether miR-27 a regulates porcine myofiber type transformation through Akt/FoxO1 signaling pathway.The results showed that overexpression of miR-27 a decreased MyHCIIa,MyHCIIx m RNA levels and slow MyHCprotein level,and increased fast MyHCprotein level.Inhibition of miR-27 a promoted the m RNA expression of all MyHCisoforms and slow MyHCprotein expression,as well as inhibited fast MyHCprotein expression.In addition,overexpression of miR-27 a decreased the level of P-FoxO1/FoxO1 and increased the level of FoxO1.However,inhibition of miR-27 a increased the level of P-Ake/Akt and decreased the level of FoxO1.Wortmannin attenuated the role of miR-27 a on porcine myofiber type transformation.These results demonstrated that miR-27 a promotes porcine myofiber type transformation form slow-twich to fast-tiwch through Akt/FoxO1 signaling pathway.Exp.5 Mi R-27 a contributes leucine-induced porcine myofiber type transformationTo examine whether miR-27 a contributes leucine-induced porcine myofiber type transformation,porcine myoblasts were transfected miR-27 a mimics followed by leucine treatment after 3 days of differentiation.We found that leucine decreased miR-27 a level,MyHCIIb m RNA level and fast MyHCprotein level.While MyHCI,MyHCIIa,MyHCIIx m RNA levels and slow MyHCprotein level were increased.Overexpression of miR-27 a attenuated the effect of leucine on porcine myofiber type transformation.These results indicated that miR-27 a contributes leucine-induced porcine myofiber type transformation.In conclusion,this study demonstrated that leucine promotes porcine myoblast differentiation and myofiber type transformation from fast-twich to slow-tiwch through Akt/FoxO1 signaling pathway.Mi R-27 a inhibits porcine myoblast differentiation and promotes porcine myofiber type transformation form slow-twich to fast-tiwch via Akt/FoxO1 signaling pathway.Mi R-27 a also contributes leucine-induced porcine myofiber type transformation.