| For mammalian,the skeletal muscle atrophy and muscle mass loss occurs in the result of unloading environment and serious stress condition,such as immovability,bed rest,weightlessness,hypoxia and low temperature.Although the hibernating mammalian,especially the small hibernating mammals,in such serious condition characterized by profound reductions in body temperature and activity,present no or only a little muscle atrophy and muscle loss during hibernation in a prolong period of immobility.What is the mechanism,which involved in the small hibernating mammals that manage to avoid muscle atrophy despite remaining stationary for long periods during hibernation? To explore the mechanism could not only provide the value new scientific information to the adaptive mechanisms in hibernating animals,but also benefit to preventing the skeletal muscle atrophy in human beings.The muscle protein loss,resulted from the protein synthesis been inhibited and the protein degradation been enhanced,is the main reason and representation of muscle mass loss commonly happened in muscle atrophy.The PI3K/AKT/mTOR signaling pathway is an intracellular signaling pathway with vital role in regulating the cell cycle and with important and developmental role in protein synthesis.mTOR controls the anabolic and catabolic signaling of skeletal muscle mass,resulting in the modulation of muscle hypertrophy and muscle wastage.Our previous findings have confirmed the function of mTOR in maintaining skeletal muscle mass.Regulating the activity of PI3K/AKT/mTOR signaling pathway to adjust the balance of protein synthesis and degradation,possible is crucial to skeletal muscle protein survival and to avoid protein loss in the skeletal muscle of hibernating mammalian.The activity of PI3K/AKT/mTOR signaling pathway is regulated by many molecules through different pathways.MicroRNAs(miRNAs),a class of endogenous non-coding small RNAs,are effective post-transcriptional regulators that modulate protein expression within a cell.Some recent researches have highlighted that some miRNAs as the essential post-transcriptional regulatory factors could affect the activity of PI3K/AKT/mTOR signaling pathway in skeletal muscle.In present study,we hypothesized that miRNAs are intimately involved in regulating the PI3K/AKT/mTOR signaling pathway activity and play significant role in avoiding the skeletal muscle atrophy in hibernating mammalian.Objectives:(1)To detect the miRNAs which could regulate the PI3K/AKT/mTOR signaling pathway activity and investigate its expression levels in the skeletal muscle of hibernating mammalian.(2)To identify the possible mechanism of miRNAs involved into the regulation on PI3K/AKT/mTOR signaling pathway to avoide the skeletal muscle atrophy in hibernating mammalian.Methods:(1)The Daurian ground squirrels(Spermophilus dauricus),a small hibernating rodent animals,were divided into six groups,Summer active group(SA),Pre-hibernation group(PRE),Early torpor group(ET),Late torpor group(LT),Interbout arousal group(IBA)and Post-hibernation group(POST).Six individuals were contained in each group.(2)The semitendinosus muscle(ST),a hindlimb skeletal muscle,was collected from the group animals in different periods of hibernation.(3)Four miRNAs,miR-206,miR-29 b,miR-21 a and miR-99 b,known to be normally expressed in skeletal muscle and can regulate the PI3K/AKT/mTOR signaling pathway,had been selected from the genomic database and the previous references.Their expression level was further analyzed through Real time-PCR in ST muscle of Daurian ground squirrels in every group.(4)The relative protein expression level of PI3 K,AKT and mTOR were measured through Western Blotting in ST muscle of Daurian ground squirrels in every group.Results:(1)The changes of body mass of Daurian ground squirrels:The body mass in summer active group was 270.67 ± 36.84 g.The body mass in pre-hibernation group was 304.30 ± 59.31 g,increasing 12%(P>0.05)than summer active group.After hibernating for two months,the individual body mass in early torpor,late torpor and inter-bout arousal group decreased 22%(P<0.01),36%(P<0.01)and 33%(P<0.05)than the individual body mass in pre-hibernation,respectively.After the whole hibernation,the individual body mass in post-hibernation had decreased 49%(P<0.001)than the individual body mass in pre-hibernation.(2)The changes of muscle mass of ST muscle: The ST muscle mass in summer active group was 0.339 ± 0.051 g.The muscle mass in pre-hibernation group was 0.374 ± 0.023 g,increasing 10%(P>0.05)than summer active group.After hibernating for two months,the muscle mass in early torpor,late torpor and inter-bout arousal group decreased 10%(P<0.05),15%(P<0.01)and 22%(P<0.01)than pre-hibernation group,respectively.While the muscle mass in post-hibernation group had decreased 28%(P<0.001)than pre-hibernation group.(3)The changes of expression level of miRNAs in ST muscle of Daurian ground squirrels in every group:a)The expression level of miR-206 in pre-hibernation,early torpor,late torpor,inter-bout arousal and post-hibernation group increased 69%(P<0.001),21%(P<0.05),15%(P>0.05),58%(P<0.001)and 98%(P<0.001)than summer active group,respectively.Compared with pre-hibernation group,the expression level in early torpor,late torpor and inter-bout arousal group decreased 28%(P<0.05),32%(P<0.05)and 7%(P>0.05),while the expression level in post-hibernation group increased 17%(P<0.05),respectively.b)The expression level of miR-29 b in pre-hibernation,early torpor,late torpor,inter-bout arousal and post-hibernation group increased 157%(P<0.001),36%(P<0.05),5%(P>0.05),94%(P<0.01)and 178%(P<0.001)than summer active group,respectively.Compared with pre-hibernation group,the expression level in early torpor,late torpor and inter-bout arousal group decreased 47%(P<0.001),59%(P<0.001)and 25%(P<0.01),while the expression level in post-hibernation group increased 8%(P>0.05),respectively.c)The expression level of miR-21 a in pre-hibernation,early torpor,late torpor,inter-bout arousal and post-hibernation group decreased 13%(P>0.05),64%(P<0.001),62%(P<0.001),36%(P<0.01)and 37%(P<0.01),respectively.Compared with pre-hibernation group,the expression level in early torpor,late torpor,inter-bout arousal and post-hibernation group decreased 59%(P<0.001),56%(P<0.001)、26%(P>0.05)and 28%(P>0.05),respectively.d)The expression levels of miR-99 b showed no significant difference among groups.The expression level in pre-hibernation,late torpor and inter-bout arousal group increased 15%(P>0.05),6%(P>0.05)and 18%(P>0.05),while the level in early torpor and post-hibernation group decreased 10%(P>0.05)and 15%(P>0.05)than summer active group,respectively.Compared with pre-hibernation group,the expression level in early torpor,late torpor and post-hibernation group decreased 22%(P>0.05),8%(P>0.05)and 26%(P>0.05),while the level in inter-bout arousal group increased 3%(P>0.05),respectively.(4)The changes of protein expression level of PI3K/AKT/mTOR in ST muscle of Daurian ground squirrels in every group:a)The protein expression levels of PI3 K showed no significant difference among groups.The expression level in pre-hibernation,early torpor and post-hibernation group decreased 13%(P>0.05),7%(P>0.05)and 4%(P>0.05),while the level in late torpor and inter-bout arousal group increased 3%(P>0.05)and 10%(P>0.05),respectively.Compared with pre-hibernation group,the expression level in early torpor,late torpor,inter-bout arousal and post-hibernation group increased 7%(P>0.05),18%(P>0.05),26%(P>0.05)and 10%(P>0.05),respectively.b)The protein expression level of AKT in pre-hibernation,early torpor,late torpor,inter-bout arousal and post-hibernation group increased 60%(P<0.001),22%(P>0.05),17%(P>0.05),73%(P<0.01)and 87%(P<0.001)than summer active group,respectively.Compared with pre-hibernation group,the expression level in early torpor and late torpor group decreased 24%(P<0.05)and 27%(P<0.05),while the level in inter-bout arousal and post-hibernation group increased 8%(P>0.05)and 17%(P<0.05),respectively.c)The protein expression level of mTOR in pre-hibernation,early torpor,late torpor,inter-bout arousal and post-hibernation group increased 123%(P<0.001),69%(P<0.01),18%(P<0.05)、135%(P<0.001)and 100%(P<0.001)than summer active group,respectively.Compared with pre-hibernation group,the expression level in early torpor,late torpor and post-hibernation group decreased 24%(P<0.05),47%(P<0.01)and 10%(P>0.05),while the level in inter-bout arousal group increased 5%(P>0.05),respectively.Discussion:(1)The PI3K/AKT/mTOR is an intracellular signaling pathway with vital role in up-regulating the protein synthesis.The findings in present study suggested:a)The highest protein expression level of mTOR in ST muscle of Daurian ground squirrels presented in pre-hibernation.And then the level decreased during hibernation,but still higher than that in summer active.The rich mTOR in ST muscle cell could enhance the protein synthesis.The increasing level of mTOR can enhance the synthesis of protein.Before and during hibernation,the relative high level of mTOR maybe benefit for maintaining the protein synthesis to counter the protein loss resulted from the torpor immovability.Such results indicate that raising mTOR content maybe is the key mechanism to preventing the skeletal muscle atrophy in mammalian hibernation.b)In pre-hibernation,the content of AKT raised a little higher than that in summer active.During hibernation,the content sustained increasing to significantly higher than summer active in post-hibernation.As the upstream of mTOR,the rising of AKT content is the possible factor to promote the expression of mTOR.The enough rich in AKT content is a probable guarantee for maintaining the high mTOR level during hibernation.c)PI3K is the upstream promoter of AKT/mTOR.In present study,there is no obvious change in PI3 K content among different periods.It maybe indicates that the PI3 K isn’t involved in the regulation on AKT and mTOR in mammalian hibernation.There possibly is another signal,not PI3 K,induces the rising of AKT and mTOR.(2)All the selected miRNAs in present study,miR-206,miR-29 b,miR-21 a and miR-99 b,are the upregulator on the PI3K/AKT/mTOR pathway.During hibernation array,the relative expression of miR-206 and miR-29 b significantly increased,and the expression of miR-99 b raised with no significance than that in summer active,while the expression of miR-21 a decreased or no obvious change.a)miR-206 is a muscle-specific miRNAs.The PI3k/Akt/mTOR as the downstream can be promoted by this miRNAs.In our data analysis showed the results of miR-206 which is highly expressed in the ST muscle during hibernation.It maybe has a great boosting effect on PI3k/Akt/mTOR pathway.b)miR-29 b has been proved as one of the upregulated miRNAs on this PI3K/AKT/mTOR pathway in skeletal muscles.PI3K/AKT/mTOR activated by miR-29 b can stimulate the protein synthesis and can play a vital role in muscle preservation throughout hibernation.c)miR-21 a is a novel myogenic miRNAs that is involved in skeletal muscle development and regulates PI3K/Akt/mTOR signaling by targeting the TGFβI gene/protein.The results showed the levels of miR-21 a were reduced or no obvious variance at multiple stages of hibernation.The evidence we provided in this study suggests that the miR-21 a possibly are not involved in the regulation on the pathway during hibernation.d)miR-99 b could upregulate the protein expression of PI3K/Akt/mTOR pathway.But miR-99 b was not significantly change during whole period of hibernation.It maybe indicates that the miR-99 b don’t give its regulation effects on the pathway during hibernation.Conclusion:This is the first study on the mechanism of miRNAs involved in preventing skeletal muscle atrophy in hibernating mammalian.The PI3K/AKT/mTOR signals are the centre regulator moleculars to control the balance between protein synthesis and degradation in mammalian skeletal muscle cell.The present study paid attention to the proteins expression of this signaling pathway in different periods of hibernation.Meanwhile,the relative expression levels of four miRNAs in ST muscle were measured in different periods of hibernation,including miR-206,miR-29 b,miR-21 a and miR-99 b,which had been reported as the regulators of PI3K/AKT/mTOR signaling pathway.Overall our finding suggests that the miRNAs(miR-206 and miR-29b)can take part in the activation of PI3K/AKT/mTOR signaling pathway to enhance the protein synthesis in skeletal muscle during mammalian hibernation.The miRNAs could play a vital role in the preserving skeletal muscle and reducing the skeletal muscle mass loss during hibernation. |
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