Study On The Expression Of MicroRNA In Rheumatic Heart Disease Patients With Atrial Fibrillation And Analysis Of The Target Genes Of Hsa-miR-145-5p And Its Function

Objective We study the expression level of micro RNA in atrial fibrillation(AF) patients with rheumatic heart disease(RHD), screen possible micro RNAs which may lead to the incidence of AF and investigate its downstream target genes and functional regulation related to AF or myocardial fibrosis, to explain the role of micro RNAs in the pathogenesis of AF, lay the foundation for further study, and also may provide new targets for seeking safe, effective drug to treat AF.Methods(1) After informed and consent, collected the atrial tissue in right atrial appendage from the patients undergoing cardiac valve replacement with RHD or valvular heart diseases, and divided them into 5 groups: ①RHD with AF group; ② RHD without AF group; ③RHD with AF group + RHD without AF group(referred to as RHD group); ④the valve disease group; ⑤RHD without AF group + valve disease group(referred to as non AF group).(2) Total RNA was extracted from the right atrial appendage, we use Affymetrix micro RNA microarray analysis to test the expression of micro RNAs in RHD patients with or without AF and in patients without RHD.(3) Real-time PCR was used to verify the micro RNAs(hsa-mi R-145-5p, hsa-mi R-1243) expression with significant difference.(4) Combined with literature searching, bioinformatics prediction software Target Scan, micro RNA.org, mi RDB were applied to predict the downstream target genes of micro RNA(has-mi R-145-5p) to search new target genes associated with AF and understand its function and mechanism.Result(1) Affymetrix micro RNA chip test results : compared with the RHD without AF group, 6 micro RNAs expression level were up regulated in RHD with AF group,while 16 down regulated, and hsa-mi R-1243 was significantly increased, hsa-mi R-145-5p was significantly down regulated; compared with the valve disease group, 46 micro RNAs expression were up regulated in RHD group, while 21 down regulated.(2) Real-time PCR results: the expression level of hsa-mi R-145-5p in RHD with AF group was significantly lower than that in RHD without AF group(P<0.01); compared with non AF group, the expression level of hsa-mi R-145-5p was significantly lower in RHD with AF group(P<0.05); there were no difference in the expression level of hsa-mi R-145-5p between RHD group and the valve disease group(P>0.05).There were no significant difference in the expression level of hsa-mi R-1243 between RHD with AF group and RHD without AF group, or between RHD with AF group and non AF group, or between RHD group and the valve disease group(P> 0.05 respectively).(3) Micro RNA target genes prediction results: there were 227 target genes of has-mi R-145-5p by the combined prediction of Target Scan, micro RNA.org and mi RDB software, among them 19 genes were related to fibrosis, extracellular matrix synthesis and ion channel regulation; by access to "human gene function manual" and America NCBI website literature, eight target genes TNFRSF11 B, TGFBR2, ADAM17, FGF5, KCNA4, CACNA1 D, MYO6, ADAMTS5 were determinate to correlate with AF.Conclusion(1) There were significant difference in the expression level of micro RNAs in AF patients with RHD, especially hsa-mi R-145-5p, hsa-mi R-1243.(2) By clinical large sample verification, the expression level of hsa-mi R-145-5p was significantly down regulated while there were no significant difference in hsa-mi R-1243.(3) hsa-mi R-145-5p may lead to AF by regulating the expression of target gene TNFRSF11 B, TGFBR2, ADAM17, FGF5, MYO6, ADAMTS5 which affect fibroblasts proliferation and collagen synthesis or by regulating KCNA4, CACNA1 D in atrial electrical remodeling.(4) hsa-mi R-145-5p can be used as marker of atrial fibrillation and new target of its drug treatment.