The Function And Mechanism Of H19/miR-675 In Weightlessness-induced Muscle Atrophy

Purpose: To observe the differentially expressed lnc RNAs in muscle in weightlessness-induced muscle atrophy model using deep sequencing;to analyze the expression of lncRNA H19 in muscle atrophy models and to explore its effect on muscle atrophy and further to clarify its mechanism.Methods: In first part the in vivo and in vitro muscle atrophy models were established using C57BL/6 and C2C12 cell line and real-time PCR,Western Blot,and immunofluorescence were used for verification.The differentially expressed lnc RNAs in soleus following 7-day hindlimb unloading were screened by deep sequencing and verified by real-time PCR.In part two,overexpressed or silenced H19 while up/down-regulated mi R-675 in myotubes at the same time followed by starvation.Real-time PCR was used to detect the m RNA expression of Atrogin-1 and Mu RF1.Western Blot was used to detect the expression of IGF1R/Akt/FOXO3 a,and the changes in myotube diameter were observed by immunofluorescence;in third part,miR-675-3p Agomi R and Antagomi R were administered into gastrocnemius through injecion,and the IGF1R/Akt/FOXO3 a pathway proteins were determined by Western Blot.Muscle cross section area and fiber number were observed by immunofluorescence.For part four the expression of let-7 family members in atrophic myotubes was measured using real-time PCR,and overexpressed let-7 and/or H19 levels to observe the interaction between them.Results:(1)The m RNA and protein levels of Atrogin-1 and Mu RF1(P<0.01 or P<0.05)were significantly increased after 7-day hindlimb unloading.Myotube starvation could up-regulate the m RNA of Atrogin-1(P<0.01),accompanied by a significant decrease in myotube diameter(P<0.01);Deep sequencing data revealed that 66 lnc RNAs were upregulated and 99 lnc RNAs were downregulated in soleus after 7-day hindlimb unloading.Among all the decreased lnc RNAs,H19 was significantly reduced as well(P<0.01).(2)The m RNA level of Atrogin-1 were significantly up-regulated after H19 overexpression(*P<0.05),and this effect could be reversed by inhibition of mi R-675 function,and overexpression of mi R-675 alone could also cause a significant upregulation in m RNA levels of Atrogin-1 and Mu RF1(P<0.01 or P<0.05)(3)Overexpression of mi R-675 down-regulated the protein expression of IGF1 R and the phosphorylation level of Akt(P<0.01 or P<0.05),and then increased the protein level of FOXO3 a and Atrogin-1(P<0.01 or P<0.05),causing a decrease in myotube diameter,muscle cross section area and fiber numbers(P<0.01 or P<0.05).The inhibition of mi R-675 caused opposite changes;(4)The RNA levels of some let-7 family members significantly increased during myotube atrophy(P<0.01 or P<0.05)and H19 overexpression effected the RNA levels of some let-7 members(P<0.01 or P<0.05).However,let-7 overexpression failed to change the RNA level of H19,Atrogin-1 and Mu RF1.Conclusions:(1)Hindlimb unloading and myotube starvation can successfully extablish muscle atrophy model;(2)The level of H19 and its encoded mi R-675 significantly decrease in muscle atrophy in tail unloading model,and its downregulation can inhibit muscle atrophy to a certain extent,which might provides protection for body;(3)Overexpression of H19 can aggravate atrophy during myotube starvation,which maybe mediated by mi R-675 through targeting at IGF1 R and negatively affecting the downstream IGF1R/Akt/FOXO3 a pathway to participate in the regulation of muscle atrophy;(4)Let-7 cannot regulate the level of lncRNA H19 in the myotube atrophy.The reason for the change of lncRNA H19 in weightless muscle atrophy needs further study.