| Objective:To establish a chronic intermittent hypoxic atrial fibrosis model,analyze the lnc RNA expression profile of atrial tissue by high-throughput technique,verify the reliability of deep sequencing results by RT-PCR,and conduct biological analysis to provide new ideas and directions for upstream treatment of atrial fibrosis.Methods:(1)Establish a chronic intermittent hypoxia atrial fibrosis model in rats Forty SD rats were randomly divided into control group(group C)and intermittent hypoxia group(group CIH).Rat atrial fibrosis model was established by continuous intermittent hypoxia for 4 weeks in CIH group.After the modeling was completed,the rats were subjected to echocardiography.Blood pressure and electrocardiogram data were measured by hemodynamics.The atrial tissue of rats was obtained,and the arrangement and size of myocardial fibers in the model group and the control group were observed by HE staining.The changes of collagen content in the atrial tissue of the control group and the control group were compared by Masson staining.Immunohistochemistry and Western blot were used to compare the changes of fibrosis-related factors(type ?collagen,type ? collagen,CTGF,TGF-?1)between the two groups.(2)Lnc RNA expression profile of central atrial tissue in chronic intermittent hypoxia model High-throughput sequencing technology was used to detect the expression profile of lnc RNA in 9 pairs of atrial tissues,screen differentially expressed lnc RNA,and differentially expressed lnc RNAs is shown by volcano maps and cluster maps.Five differentially expressed lnc RNAs(NONRATT011456.2,NONRATT013797.2,NONRATT033351.1,NONRATT001596.2,NONRATT016396.2)were randomly selected for RT-PCT experiments to verify the accuracy of high-throughput sequencing.The potential function of differentially expressed lnc RNAs was predicted by GO analysis and KEGG enrichment analysis.The lnc RNA-related mi RNAs and m RNAs were predicted using databases such as "mi Randa" to establish a lncRNA-miRNA-mRNA network.Results:(1)After 4 weeks of chronic intermittent hypoxia,no difference in body weight or heart weight was found between the two groups.Echocardiography showed no significant difference in the left ventricular end-systolic and end-diastolic diameter,left ventricular ejection fraction and interventricular septal thickness.However,in the CIH group,the left atrial diameter and the mean pulmonary artery pressure were higher than those in the control group.There were no significant differences in blood pressure and ECG data between the two groups.(2)Compared with the control group,the CIH group showed extreme disorder of atrial myocytes and increased myocardial space.Masson staining showed a significant increase in collagen fibrosis deposition in the CIH group,and atrial tissue collagen volume fraction increased by 47% compared with the control group.Immunohistochemical and Western blot results showed that the expression of type I collagen,type III collagen,CTGF and TGF-?1 protein in atrial myocardium of CIH group increased compared with the control group.(3)Lnc RNA expression profile: According to the results of high-throughput analysis,a total of 23528 differentially expressed genes were detected in lnc RNAs,of which 457 newly predicted lnc RNAs,and the control group compared with the model group There were 285 lnc RNAs with a difference of 2 times(|log2FC|>1),and there were 120 up-regulated lnc RNAs and 165 down-regulated.The most significant difference in up-regulation of lnc RNA was NONRATT015292.2(log2FC=7.05429,P=0.010054),and the difference in lnc RNA down-regulated was most significant as NONRATT022310.2(log2FC=-7.07017,P=0.001434).(4)RT-PCR results showed that Compared with the control group,the expression of NONNATT011456.2,NONRATT013797.2,NONRATT033351.1,NONNATT016396.2 in the chronic intermittent hypoxia group was up-regulated,which was the same as the high-throughput sequencing trend.The verification results of NONRATT001596.2 have the same trend as the high-throughput sequencing results,but there is no statistical difference.It is suggested that lnc RNA high-throughput sequencing results have high reliability and accuracy.(5)These differentially expressed lnc RNAs were analyzed using biological software.GO enrichment analysis of differentially expressed lnc RNAs shows significant enrichment of the biological process are cellelur process,single-organism process,biological regulation,etc.The most prominent cellular components for enrichment are cell,cell part,organelle,etc.The most significant molecular functions of enrichment are binding,catalytic activity,and nucleic acid transcription factor activity.KEGG analysis revealed that differentially expressed lnc RNA is mainly enriched in NF-kappa B signaling pathway,TGF-?1 signaling pathway,IL-17 signaling pathway.Establishment of lnc RNA-mi RNA-m RNA co-expression network:A total of 5603 pairs of ce RNA were obtained by differential mi RNA,m RNA,and lnc RNA analysis.Conclusions:(1)Atrial fibrosis model was successfully established by chronic intermittent hypoxia,which can be used as atrial fibrosis research;(2)There were a large number of differentially expressed lnc RNAs in the atrial tissue of the rats with chronic intermittent hypoxia atrial fibrosis;(3)RT-PCR verification results and high-throughput sequencing results have good consistency,suggesting that lnc RNA high-throughput sequencing results have higher reliability and accuracy;(4)Using biological software to analyze these differences lnc RNAs may be involved in many biological processes such as TGF-?,NF-?B,and establish a co-expression network of lnc RNA-mi RNA-m RNA,suggesting that differentially expressed lnc RNA may be associated with atrial fibrillation and atrial fibrosis. |
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