A Preliminary Study On The Relationship Between The Intervention Of Aerobic Exercise On Heart Failure Rats And The MiRNA Located In Mitochondria

Background: Mitochondria are semi-autonomous cellular organelles with their own genome.It is the main site for intracellular oxidative phosphorylation and synthesis of adenosine triphosphate(ATP),providing energy for cellular activity.Micro RNAs(mi RNAs)are usually 20–24 nt long,non-coding RNAs,involved in post-transcriptional gene regulation by binding to the 3?-untranslated regions(3?-UTRs)of target m RNA,which impact on diverse cellular processes.Objectives: To determine whether aerobic exercise causes changes in mitochondrial-localized mi RNAs in a rat model of heart failure caused by post-load aerobic exercise stress,thereby mitigating heart failure by regulating energy metabolism.Methods:1.Animal model: 48 male rats(wistar)were divide into 4 groups randomly: early heart failure was exerted aerobic exercise(AT),early heart failure group(AC),healthy rats were exert aerobic exercise(ST),and negative control(SC).Abdominal aorta constriction was performed to establish heart failure model,the way of exercise was run on the aclinic treadmill 60min/day,the intensity was 20m/min,5days per week and last for 8weeks.2.Detection of ultrasonic scanning image system: System of Visual Sonics Vevo2100 was used to confirm early heart failure and the intervention of exercise after 8weeks of operation and 8 weeks of exercise training respectively.LVIDs,LVIDd,IVSs,IVSd,LVPWs and LVPWd reflected the change of left ventricular structure,EF and FS was detected to reflect functional change.3.Superpure mitochondria isolation: all rats were killed after 8 weeks exercise intervention in 72 hours.Percoll was used to separation.Western blot and capillary electrophoresis were performed to verification.4.Mitochondria RNA extraction: methods of Trizol was applied to extraction,Na Nodrop 2000 was used to detect the purity and concentration.5.RNA high-throughput sequencing: selected 3 samples randomly each group,small RNA pool was built.The results of sequencing were analyzed and settled.Classify known mi RNA and novel mi RNA.6.Bioinformatics analysis: compared with known mi RNA profiling each groups,to find out difference expression mi RNA.All novel mi RNAs were summarized and screened.To verify mi RNA structure.7.SYBR Green q RCR Master Mix.SYBR Green q RCR was utilized to detect if the H9C2 cells overexpressed Novelmi RNA-162 ? Novelmi RNA-175 ?Novelmi RNA-223?Novelmi RNA-378?Novelmi RNA-207?Novelmi RNA-362?Novelmi RNA-211?Novelmi RNA-355?Results1.High-throughput sequencing result revealed: rno-mi R-99b-5p in group ST upregulated notably compared with SC.Rno-mi R-99b-5p upregulated in AC compared with SC.Rno-mi R-450-5p,rno-mi R-16b-5p,rno-mi R-674-5p and rno-mi R-10a-5p were downregulation in AT compared with SC.2.Compared between AT and SC,8 mi RNA had significant difference,1 of them downregulated,7 of them upregulated.Compared between AT and AC,37 mi RNA had significant difference,3 of them downregulated,34 of them upregulated.Compared between AT and ST,21 mi RNA had significant difference,2 of them downregulated,19 of them upregulated.Compared between AC and ST,2 mi RNA had significant difference,1 of them downregulated,the other one upregulated.Based on secondary structure prediction software and the difference profiling in various groups.9 nucleotide sequences were pitched on: Novelmi RNA-162 ?Novelmi RNA-175?Novelmi RNA-223?Novelmi RNA-378?Novelmi RNA-207?Novelmi RNA-362 ? Novelmi RNA-211 ? Novelmi RNA-355.RT-q PCR demonstrated that cells can overxpress Novelmi RNA-162?Novelmi RNA-207?Novelmi RNA-362?Novelmi RNA-378.Conclusion1.There was a change in mitochondrial-targeted mi RNAs during aerobic exercise intervention in heart failure rats.2.RT-q PCR validated the candidate mi RNA and finally found that Novelmi RNA-162,Novelmi RNA-378,Novelmi RNA-207,and Novelmi RNA-362 may be mi RNAs that locate in mitochondria and affect heart failure.