Construction Of CircRNA Expression Profiles And Functional Analysis Of Gastrocnemius Muscle In Rats Subjected To Long-term Aerobic Exercise And Acute Exhaustive Exercis

Background:Exercise has become a part of our daily productive life,and reasonable exercise is essential to maintain a healthy state of body and to prevent or delay the occurrence and development of diseases,and even exercise therapy has become one of the important ways to treat diseases.Research on exercise science is currently in full swing,but due to the diversity and complexity of exercise,we have not been able to fully reveal the intrinsic mechanisms of exercise in the body,and the mechanisms of exercise regulation of skeletal muscle,the largest organ of the body,are extremely complex.In many cases,exercise is a modifying regulation of gene expression,of which post-transcriptional regulation is an important mode of gene expression regulation.In recent years,in the field of post-transcriptional regulation,a novel RNA molecule,circ RNA,has been shown to regulate gene expression as a mi RNA sponge.circ RNA acts as a competitive endogenous RNA at the post-transcriptional level by binding to mi RNAs and blocking their repression of target genes,thus performing important regulatory functions at the post-transcriptional level.As research continues to progress,many circ RNAs have been identified in various disease models,and they serve as potential targets to influence disease development to some extent.Then,exercise will certainly also induce changes in circ RNA expression,but the current research in this area is basically in a blank state.Therefore,we hope to construct a circ RNA expression profile in rat skeletal muscle under exercise intervention and explore the mechanism by which circ RNA regulates exercise-related protein expression through mi RNA.Research purpose:Based on the important role of circ RNAs in post-transcriptional regulation and their blank state in exercise research,we established the expression profiles of circ RNAs in skeletal muscle of rats under aerobic exercise training and one acute exhaustive exercise state,and compared them with the expression profiles of skeletal muscle circ RNAs of rats in long-term resting state,conducted network construction of differentially expressed circ RNAs,explored the possible mechanisms of the role of some circ RNAs at the theoretical level,and then provided insight into the effects of aerobic exercise on skeletal muscle,and provided new perspectives for the elucidation of health mechanisms of exercise intervention.Research methods:1.Establishment of exercise intervention rat model.Eighteen 6-week-old SD rats were randomly divided into three groups with 6 rats in each group:long-term aerobic exercise group(AE),acute exhaustive exercise group(EE)and quiet control group(C).The AE group received aerobic exercise training for 8 weeks,the EE group received acute exhaustive exercise for 8 weeks after normal feeding,and the C group received 8 weeks normal feeding.2.circ RNA expression profile of rat skeletal muscle was constructed.Three rats in each group were randomly selected to extract total RNA from gastrocnemius muscle for high-throughput sequencing to obtain circ RNA expression profiles.The sequencing results were analyzed,as well as GO hierarchical enrichment analysis and KEGG pathway enrichment analysis.3.Verify the ring formation authenticity,sequencing authenticity and intergroup expression of circ RNA.Circrna-specific Divergent Primer was designed according to the principles of circ RNA primer design.Total RNA was extracted from rat gastrocnemius muscle,and the total RNA was digested by RNase R to remove linear RNA.The circ RNA sequence was verified by RT-PCR and sanger sequencing.Using RNA reverse transcription products as template,RT-q PCR was performed to verify the up-down-regulation relationship of circ RNA among the three groups of rats.4.The target mirnas and mi RNA target genes of circ RNA were predicted by bioinformatics methods,and the interaction network diagram of Circr Na-mir Na-mrna was constructed to analyze the function of target genes.Results:1.A total of 155.66 Gb Clean Data was obtained,and 1073 circ RNAs were predicted by CIRI software and find＿circ software together,and the known circ RNAs were found to be 562 and the newly predicted circ RNAs were 511 when compared with circ Base database.The number of new circ RNAs predicted was 511.Most of the circ RNAs originated from exonic regions and were irregularly distributed on each chromosome without regular distribution,with lengths concentrated in the range of400-1000 nts.A total of 67 differentially expressed circ RNAs were predicted by twoby-two comparison between the three groups,among which,14 circ RNAs were upregulated and 29 circ RNAs were down-regulated in the AE group compared with the C group;13 circ RNAs were up-regulated and 19 circ RNAs were down-regulated in the EE group.13 circ RNAs were up-regulated in the AE group compared with the EE group and 16 circ RNAs were down-regulated.2.Among the GO-level enrichment of parental genes differentially expressing circ RNAs,biological processes were mainly enriched in actin filament organization,control of calcium channels,skeletal muscle phylogeny,alveolar development,Golgi organization,etc.Molecular functions were mainly enriched in GTP activator activity,GTP binding,DNA binding transcriptional activator activity,actin filament binding,carbohydrate binding,phospholipid KEGG pathway is mainly enriched in ECMreceptor interaction,endocytosis,lysine degradation,and transcriptional dysregulation in tumors.Here,GO and KEGG enrichment are only listed for processes and pathways that enrich for a high number of parental genes.3.Four circ RNAs,named circ Fam208 a,circ Dym,circ Ash1 l and circ Ralgapa1,were successfully validated by RT-PCR experiments and RNase R digestion experiments.the differential expression of these four circ RNAs among three groups of rats was verified by RT-q PCR experiments,and the results were consistent with the sequencing results The results were consistent with the sequencing results.4.Using bioinformatics software analysis,a ce RNA interaction network map was constructed around circ Fam208 a,circ Dym,circ Ash1 l and circ Ralgapa1.The four circ RNAs bound to a total of 37 mi RNAs and targeted to 340 m RNAs,respectively.Conclusions:The expression profile of circ RNA in rat skeletal muscle was successfully constructed,and 67 differentially expressed Circrnas were predicted.The differentially expressed circ RNA parental genes were mainly enriched in functions related to muscle contraction,energy metabolism,oxidative stress,inflammation,signal transduction and so on.It provides data support for the follow-up study.