| ObjectiveThe objective of this study is to screen and identify of differentially expressed miRNA in rats with atrial fibrosis based on high-throughput sequencing technology,thus providing potential targets for the diagnosis and treatment of atrial fibrosis.MethodsForty male SD rats were randomly divided into the chronic intermittent hypoxia model group and the control group.The rats in the model group underwent chronic intermittent hypoxia by using technical hypoxia device.After 4 weeks,echocardiography and hemodynamic examination were performed,relevant parameters were recorded.HE staining,Masson staining,IHC assay and Western Blot were performed to evaluate atrial fibrosis.Atrial samples from both groups were sequenced by high-throughput sequencing technology.Bioinformatics analysis were performed to find out their potential functions.Among those differentially expressed miRNA and m RNA,miR-29b-3p and miR-29b-5p were selected for verification by using the technology of RT-qPCR.The level of wnt2 b m RNA and wnt2 b protein expressed in both group was detected by RT-qPCR and Western Blot respectively.Results1.As the echocardiography revealed,contrast to the control group,LAD and m PAP were all increased in model group(p<0.05).Cardiac Ultrasonic analysis of rat heart showed decreased cardiac systolic function.2.In comparision with the control group,significant morphology and structure disorder of atrial cardiomyocytes and the myocardial interstitial fibrosis were observed.The CVF of atrial tissue in model group increased 216.3%(p<0.001).Moreover,the protein expression levels of col-1,col-3,CTGF,TGF-β1 in the model group were significantly increased by 304.4%(p<0.001),442.5%(p<0.001),220.6%(p=0.01),and 159.5%(p<0.001)respectively than that of the control group.3.By means of high-throughput sequencing technology,a total of 1204 differentially expressed miRNA were sceened out,of which 615 miRNAs were newly predicted.20 differentially expressed miRNAs were screened out,including 12up-regulated miRNAs and 8 down-regulated miRNAs,while 436 differentially expressed m RNAs was screened out,with 381 m RNAs up-regulated and 55 m RNAs down-regulated.Real-time PCR verified that the expression levels of miR-29b-3p and miR-29b-5p were statistically down-regulated in the model group,which was consistent with high-throughput sequencing results.4.GO analysis showed that the target gene of these miRNAs were enriched in biological regulation,cytoplasm,nucleus,molecular function,protein binding,etc;differentially expressed m RNAs were significantly enriched in biological regulation,stress response,binding,etc.KEGG analysis showed that the miRNA were mainly enriched in the MAPK signaling pathway;the differentially expressed m RNA was significantly enriched in cardiovascular disease,translation,signal transduction,etc.5.The expression levels of wnt2 b m RNA and wnt2 b protein in the model group were both significantly increased(p<0.05)in sharp contrast to the control group.ConclusionsThe atrial fibrosis model can be successfully established in the way of chronic intermittent hypoxia.The expression profile of differetially expressed miRNA and m RNA among the chronic intermittent hypoxia model group and the control group can be sceened and preliminarily established by High-throughput sequencing technology.Those differentially expressed miRNA and m RNA is of great significance for exploring the pathological mechanism involved in atrial fibrosis..Real-Time PCR verified that mir-29b-3p and mir-29b-5p were statitically down-regulated in the chronic intermittent hypoxia model group,which was consistent with the results of high-throughput sequencing,suggesting that miR-29 may be involved in the pathogenesis of atrial fibrosis.The transcription and translation levels of wnt2 b were increased significantly in the chronic intermittent hypoxia model group,suggesting that wnt2 b may be involved in the occurrence and development of atrial fibrosis. |
