Molecular Mechanism Of Long Non-coding RNA SYISL Promoting Muscle Atrophy

Skeletal muscle accounts for about 30%-50% of the total body weight of animals.It plays a very important role in a variety of life activities,such as limb and trunk movements,the overall metabolism of animals,and the storage of body proteins.The integrity of skeletal muscle is the key factor for its normal metabolism and biological function.Skeletal muscle metabolism usually includes both protein bio-synthesis and degradation.When protein synthesis decreases or protein degradation increases,metabolic abnormalities can lead to a decrease in the protein content of the body’s skeletal muscle,making it difficult for the muscle to perform its normal biological functions,leading to a decrease in muscle production and quality damage,affecting the development of animal husbandry and human health.Lnc RNA(Long noncoding RNA,Lnc RNA)is a kind of RNA with transcript length of more than 200 nt and no protein-coding ability.Lnc RNA can participate in a variety of life processes and play an important role.For example,lnc RNA can participate in the process of muscle development,muscle hypertrophy and muscle atrophy.SYISL is a lnc RNA located in the SYNPO2 intron identified by our laboratory in the previous results.The previous results showed that SYISL could promote cell proliferation,fusion but inhibit the differentiation of muscle cells by recruiting PRC2 complex at the stage of muscle growth and development.With the aging of the body,SYISL can accelerate the muscle atrophy process which was induced by aging and aggravate the muscle atrophy degree of 3-month-old mice induced by DEX.Furthermore,it was further verified that SYISL was highly conserved in mice and pigs.Based on our previous research,this study further explores the molecular mechanism of SYISL promoting muscle atrophy and whether its human SYISL promotes skeletal muscle atrophy through the same molecular mechanism.1.The position conservation of human,pig and mouse SYISL was analyzed by UCSC database.The results showed that there was a transcript AK238214(p SYISL)on pig chromosome 8 transcribed by the first intron of SYNPO2,and a transcript AK021986(h SYISL)on human chromosome 4 transcribed by the fourth intron of SYNPO2;Sequence conservation analysis showed that these three lnc RNAs had low sequence conservation.The results of nucleocytoplasmic separation and RNA FISH showed that h SYISL is expressed in both the nucleus and cytoplasm,and the expression level in the cytoplasm is slightly higher than that in the nucleus,and p SYISL mainly distributed in the nucleus.2.Through the prediction of mi RNA binding sites,the verification of dual luciferase report experiments,q RT-PCR,Western blot,and other experiments,we found that mi R-103-3p,mi R-23a-3p,and mi R-205-5p could target with SYISL,p SYISL,and h SYISL to reduce their dual luciferase activities.Overexpression of these three mi RNAs in C2C12 cells can reduce the expression level of SYISL,indicating that SYISL may play a role through these three mi RNAs.3.The expression of mi R-103-3p,mi R-23a-3p,and mi R-205-5p in skeletal muscle of mice at different ages was detected by q RT-PCR.It was found that the expression of mi R-23a-3p gradually decreased with the aging of mice,and the expression of mi R-103-3p was relatively stable,while the expression of mi R-205-5p was the peak at the age of 12 months.The expression changes of mi R-103-3p,mi R-23a-3p,and mi R-205-5p in different muscle atrophy models were detected.In various muscle atrophy models,the expression of mi R-103-3p and mi R-23a-3p were significantly down-regulated,and the expression of mi R-205-5p m RNA was significantly up-regulated.4.Through q RT-PCR,Western blot,RNA-pulldown,and dual luciferase detection experiments,it is verified that mi R-23a-3p can significantly reduce the fluorescence activity of Luc-Mu RF1-3’UTR and Luc-Atrogin-1-3’UTR,while SYISL can competitively bind mi R-23a-3p with Mu RF1-3’UTR and Atrogin-1-3’UTR.After overexpression of SYISL-Mut,the expression of Mu RF1 and Fox O3 a was still significantly up-regulated,while the expression of Atrogin-1 at m RNA and protein levels had no significant change.It shows that SYISL can promote muscle atrophy by acting as a molecular sponge of mi R-23a-3p,and there are other molecular mechanisms to promote muscle atrophy.5.Verify the combination of mi R-103-3p with Mu RF1,mi R-205-5p with Mu RF1,and Fox O3 a through RNAhybrid 2.12 prediction and dual luciferase report experiments;The effect of mi R-103-3p and mi R-205-5p on muscle atrophy were detected in C2C12 cells by cell transfection,q RT-PCR,Western blot,etc.The results showed that mi R-103-3p/mi R-205-5p can inhibit muscle atrophy.6.Through lentivirus packaging,mice leg muscle lentivirus injection,q RT-PCR,Western blot,dual luciferase report,and other experiments,further verify that SYISL can as a molecular sponge of mi R-23a-3p/mi R-103-3p/mi R-205-5p promote muscle atrophy.7.Through lentivirus packaging,mice leg muscle lentivirus injection,q RT-PCR,Western blot,and other experiments,the changes of muscle after overexpression of h SYISL in DEX-induced mouse muscle atrophy model and aging induced muscle atrophy model were verified.The results showed that overexpression of h SYISL could aggravate these two types of muscle atrophy.h SYISL has the function of promoting muscle atrophy.The dual luciferase report experiments further verified that h SYISL can act as a molecular sponge of mi R-23a-3p/mi R-103-3p/mi R-205-5p promoting muscle atrophy.In conclusion,our studies further explored the molecular mechanism of SYISL promoting muscle atrophy.SYISL can act as a molecular sponge of mi R-23a-3p/mi R-103-3p/mi R-205-5p promotes muscle atrophy.Our study provides an important theoretical reference for clarifying the molecular mechanism of lnc RNA regulating muscle atrophy,the healthy development of animal husbandry,and the treatment of human diseases.