| Background: Heart failure is a serious public health problem in China.Aerobic exercise interventions have a positive effect on preventing heart failure and delaying heart failure,but the current mechanism is unclear.In the previous work,we obtained a rat model of heart failure adopting abdominal aortic coarctation and performed an aerobic exercise intervention for 8 weeks.The results showed that aerobic exercise altered the mi RNA expression profile of cardiomyocytes and enhanced mitochondrial function.We consider that the changes in mi RNAs expression regulate cardiac function-related target gene expression,leading to mitochondrial dysfunction and structural abnormalities,thereby delaying heart failure progression.Cytoplasmic localized mi RNAs and mitochondria-localized mi RNAs work together in this process.However,the study of mitochondria-localized mi RNA(mitomi R)is currently not carried out,and its expression profile in rat myocardium is unclear,and aerobic exercise can change the localization of mi RNA.To investigate the role of mitomi R in aerobic exercise intervention in heart failure,we isolated ultrapure mitochondria from left ventricular myocardium in different treatment groups and extracted mitochondrial RNA for high-throughput sequencing.Sequencing results showed that aerobic exercise had a significant effect on the change of mitomi R expression profile.Among them,in addition to changes in expression levels or localization of some known mi RNAs,we also obtained 412 first discovered nucleic acid sequences localized in the mitochondria.The 412 nucleic acid sequences were fitted to the secondary structure by bioinformatics methods,and 44 sequences conforming to the secondary structure of the mi RNA were obtained.We believe that there must be some of the first discovered mitomi R,which plays an important role in the aerobic exercise intervention in heart failure.Research purposes: To verify the presence of new mitomi Rs in 44 pre-worked candidate mi RNA sequences that match the mi RNA secondary structure.To search for new target genes for mitomi R,explore the regulation of mitomi R on mitochondrial biosynthesis and functional recovery,and then discuss its role in aerobic exercise and treatment of heart failure,and provide theoretical basis and possible intervention targets for rehabilitation and treatment of heart failure.Research methods: 1.Design of primers: mi RNA primers are slightly different from common m RNA primers,and specific reverse transcription primers need to be designed to extend the sequence length of c DNA to meet the needs of subsequent experiments.Therefore,all mi RNA primers required for this experiment were designed according to the micro RNA RT-PCR primer design method.2.Verification of mitomi R: Total RNA was extracted by Trizol method,and whether the candidate mi RNA sequences selected by RT-q PCR were verified to have transcripts in the cells or not.3.Validation of candidate target genes: The EGFP fluorescence reporter system was used as a means(insertion of the mitomi R-binding fragment of the candidate target gene in the 3'UTR region of the EGFP expression plasmid to detect the expression level of EGFP)to determine the regulation of mitomi R on the expression of candidate target genes.The pc DNA-EGFP vector was digested with Bam HI and Eco RI to construct a recombinant plasmid for predicting the target site,and the pc DNA3-U6M2 vector was digested with Pme I and Xba I to construct a mitomi R-207 expression plasmid.The recombinant plasmid pc DNA3-U6M2-mitomi R-207 was overexpressed in H9C2 cardiomyocytes,and transfected with empty vector as control to determine the transfection efficiency of pc DNA3-U6M2-mitomi R-207;co-transfection in HEK-293 T human embryonic kidney epithelial cells pc DNA3-U6M2-mitomi R-207,pc DNA3-target gene sequence-EGFP recombinant plasmid and ps D-Red-N1 plasmid were used to verify whether mitomi R-207 regulates the expression of a candidate target gene.Results: 1.The 44 candidate mi RNA sequences selected in the previous experiments were sequenced and sequenced into 26 sequences.The 26 candidate mi RNA sequences were verified by RT-q PCR,and 15 of them were amplified.2.The PCR products of the 15 sequences were sequenced.The which sequencing results of the amplified products with 9 sequences were consistent with those of the previous deep sequencing,tentatively set as the new mitomi R.They are novel miRNA-6,novel mi RNA-50,novel mi RNA-64,novel mi RNA-98,novel mi RNA-115,novel mi RNA-207,novel mi RNA-275,novel mi RNA-291 and novel mi RNA-3053.We selected a mitomi R with high expression level and the greatest change in aerobic exercise intervention for further study,namely novell mi RNA-207 which is mi RNA-207 in the 9 above.The results showed that mitomi R-207 is a dual-localized mi RNA that targets both mitochondrial regulation of mitochondrial genome expression and regulation of nuclear-encoding gene expression in the cytoplasm.4.mitomi R-207 has 13 possible binding sites on rat mitochondrial genome,which is a possible regulatory target by bioinformatics predicts.The binding sites are distributed in ND4,ND5,ND6,COX1,COX3,Trnd,Trnk(three binding sites),RNr-2,and RNr-1(two binding sites).5.The results of the EGFP fluorescence reporter system showed that mitomi R-207 at least can up-regulate the expression of mitochondrial genes ND4 and ND6.Conclusion: mitomi R-207 is a newly discovered mi RNA that localizes both in the cytoplasm and in the mitochondria.In the process of aerobic exercise intervention for heart failure,mitomi R-207 is highly expressed in mitochondria,up-regulating the expression levels of mitochondrial genes ND4 and ND6. |
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