The Expression And Validation Of MicroRNA-520d-5p In Atrial Tissue Of Rheumatic Heart Disease Patients With Atrial Fibrillation And Its Target Gene Prediction Research

Objection To explore the expression differences of the miR-520d-5p in atrial tissue in rheumatic heart disease patients with atrial fibrillation and predict its possible downstream target genes, to seek for the safe and effective drugs to provide new targets for the treatment of atrial fibrillation. Methods(1)By the informed consent, gather right auricle myocardial tissues of rheumatic and valvular disease patients with valve replacement, which is divided into 5 groups: ① rheumatic merger atrial fibrillation group(referred to as rheumatic atrial fibrillation group);② rheumatic without atrial fibrillation group( referred to as rheumatic without atrial fibrillation group);③ the rheumatic heart valve disease group(referred to as valvular disease group);④ rheumatic atrial fibrillation group + rheumatic without atrial fibrillation group(referred to as rheumatic group);⑤the rheumatic without af + valvular disease group(referred to as no atrial fibrillation group);(2)after extract the right auricle organization total RNA and qualified quality inspection, applicate Affymetrix mirnas chip to test and analyse microRNAs expression differences of rheumatic atrial fibrillation group 、rheumatic without atrial fibrillation group、no atrial fibrillation group, filter out significant expresse difference of microRNAs.(3) by using rt-pcr method to screen out the purpose of miRNAs(hsa-miRNA-520 d-5p and hsa-miRNA-4723) and verify them in large sample size of the organization;(4) applicate bioinformatics software(TargetScan, mi RDB and microRNA.org) to predict hsa- miRNA-520d-5p downstream target genes, and combined with the function of the human genome’s handbook and NCBI platform to filter out target genes associated with atrial fibrillation and understand the possible mechanism of their function. Results(1) Affymetrix mirnas chip test results: rheumatic atrial fibrillation group compared with rheumatic without atrial fibrillation group, six microRNAs expression upregulateed, 16 miRNAs expression downregulateed, which hsa-miR-4723 upregulateed obviously, hsa-mi R-520d-5p obviously downregulateed;Rheumatic atrial fibrillation group compared with non rheumatic group, 46 microRNAs expression upregulateed, 21 expression downregulateed; rheumatic atrial fibrillation group internal comparision(A1069 sample vs A1070 samples), 196 microRNAs expression upregulateed, 234 microRNAs downregulateed.(2) the Real-time PCR verification results:(1) hsa-miR- 520d-5p expression abundance in rheumatic atrial fibrillation group significantly lowered, rheumatic atrial fibrillation group had significant statistic difference compared with rheumatic wothout atrial fibrillation group(P < 0.05), and compared with no atrial fibrillation group also have significant statistic differences(P < 0.05);And rheumatic group compared with non rheumatic group,hsa-miR-520d-5p expression abundance difference has no statistical significance differences(P > 0.05).(3) mirna target genes predicte results: TargetScan, microRNA.org, miRDB common prediction for hsa-miR-520d-5p have 491 target genes, among which 39 may be closely related to signal transduction pathways, myocardial fibrosis, collagen synthesis, finally 9 target genes associated with atrial fibrillation: ADAM10/15/17, SMAD3, TEAD-1, COL1a1/3 a1, Zeb2 Meox2, DLG5 and PDE2, MEF2 A and TGFβ. Conclusion(1) atrial organization microRNAs expression profile chip of the rheumatic heart disease patients with atrial fibrillation is obviously abnomal.(2) the clinical large sample verification, atrial fibrillation atrial tissue hsa-miR-520d-5p expression levels significantly lower, and hsa-miR-4723 expression level has no obvious change.(3) hsa-miR-520d-5p may participate in the occurrence of atrial fibrillation and maintain through regulating ADAM10/15/17, COL1a1/3, SMAD3, TEAD- 1 a1, Zeb2 Meox2, DLG5 and PDE2, MEF2 A and TGF β target gene expression,impacting the signaling pathways, collagen metabolism, myocardial fiber cells to participate in the occurrence and maintaining of atrial fibrillation.