3.1 Baseline characteristics of the subjects
The baseline demographic characteristics of the three cohorts were shown in Online Table1 to Table3. The distributed characteristics were similar between the two groups. 30 paroxysmal AF and 30 persistent AF received radiofrequency catheter ablation in the second cohort.
3.2 Plasma miRNAs level in atrial fibrillation
The top 25 downregulated and upregulated differences in miRNAs expression between the two groups were illustrated with a heatmap (Figure 1A). The screening criteria indicated that, 4 miRNAs (hsa-miR-302, hsa-miR-193a-5p, hsa-miR-153-3p, hsa-miR-551-3p) were upregulated in the AF group, while 6 miRNAs (hsa-miR-199-3p, hsa-miR-1260, hsa-miR-144-3p, hsa-miR-425-5p, hsa-miR-21, hsa-miR-22-3p) were downregulated (Table 1).
3.3 Comparing the qRT-PCR and miRNAs sequencing
The miR-199-3p and miR-22-3p were studied in our previous study. And miR-21 were demonstrated positively associated with atrial fibrosis in AF in other study . The results demonstrated that hsa-miR-302, hsa-miR-193a-5p, hsa-miR-153-3p, and hsa-miR-551-3p were upregulated, whereas hsa-miR-1260, hsa-miR-144-3p, hsa-miR-425-5p were downregulated. These results were similar to the miRNA sequencing results (Figure1B-C).
3.4 Verification of miRNAs
7 candidate miRNAs were detected in an independent cohort of 80 AF patients and 80 healthy controls by qRT-PCR. The miR-144-3p and miR-425-5p were downregulated in the AF group whereas hsa-miR-302 was up-regulated. This was consistent with the results in the first miRNA sequencing cohort. The expression of the other 4 miRNAs was inconsistent with the sequencing results (Figure 2A-C). We found that the expression of miR-144-3p and miR-425-5p was downregulated in left atrial blood than in the peripheral blood. On the contrary, expression of miR-302 was upregulated in the left atrial blood than in the peripheral blood (Online Figure 1).
3.5 miRNAs expression in atrial tissue in AF patients and mice AF model
We established that the expression of miR-144-3p and miR-425-5p was decreased in the AF than in the SR whereas miR-302 was increased (Figure 2D-E). This was in agreement with the plasma results. We also realized that the expression of miR-144-3p and miR-425-5p was lower in the left atrial tissue compared to the right atrial tissue, and miR-302 expression was increased in the left atrium tissue in mouse AF model (Online Figure 2). Therefore, these findings implied that the expression level of miR-144-3p and miR-425-5p was lowered, while that of miR-302 was increased in left atrial in comparison to the right atrial tissue in AF.
3.6 miRNAs expression in different type of AF
To detect whether the differentially expressed miRNA was associated with AF type, the miR-302, miR-144-3p, and miR-425-5p were detected in 40 paroxysmal AF and 40 persistent AF, and results showed the expression level of miR-144-3p and miR-425-5p was lower in persistent AF than in paroxysmal AF (Figures 2F-G). However, miR-302 expression difference was not significantly between the two different types of AF (Figure 2H).
3.7 Diagnostic value of the miRNAs for AF
ROC analyses were performed on the AF and healthy individuals. All the three miRNAs could differentiate AF from healthy control with AUC of 0.928 (95% CI: 0.885-0.970, 88% sensitivity and 89% specificity), for miR-144-3p, 0.921 (95% CI: 0.881-0.961, 95% sensitivity and 77% specificity) for miR-425-5p, 0.891 (95% CI: 0.838-0.945, 86% sensitivity and 81% specificity) for miR-302, respectively (Fig3A). Considering the difference expression of miRNAs in paroxysmal and persistent AF, ROC curve analysis was carried out in paroxysmal and persistent AF, and the AUC was 0.906 (95% CI: 0.842-0.969, 73% sensitivity and 99% specificity ) of miR-144-3p, 0.888 (95% CI: 0.817-0.958, 85% sensitivity and 85% specificity ) of miR-425-5p and 0.681 (95% CI: 0.564-0.798, 45% sensitivity and 83% specificity ) of miR-302 (Fig 3B).
3.7 Plasma level of miR-144-3p and miR-425-5p was negatively correlated with the left atrial fibrosis in persistent AF
The left atrial fibrosis was evaluated using the extent of left atrial low voltage area (LVA) by left atrial voltage matrix mapping during radiofrequency catheter ablation in the persistent AF. The plasma miR-144-3p and miR-425-5p level were negatively associated with the left atrial fibrosis by Pearson’s correlation coefficients (Fig4A-B). However, the plasma miR-302 level was not found to be linked to the left atrial fibrosis (Fig4C).
We divided the left atrial low voltage area into 3 groups according to the extent of left atrial fibrosis (group 1: LVA extent between 0% to 10%, group 2: LVA extent between 10% to 20%, group 3: LVA extent between over 20%), and the results showed that the expression levels of miR-144-3p and miR-425-5p were lowest in group 3 and the highest in the group1 (Online Figure 3).
3.8 Comparison of plasma miRNA expression level in the pre-RFCA and post-RFCA
The plasma miR-144-3p and miR-425-5p level were detected in 20 AF patients during pre-RFCA (radiofrequency catheter ablation) and post-RFCA, and also compared with the control group. The relative plasma level of miR-144-3p increased in post-RFCA compared with pre-RFCA (the median and inter-quartile ranges of pre-RFCA vs. post-RFCA were 1.10:0.34 and 0.762:0.2963, respectively). The level of miR-425-5p also increased (the median and inter-quartile ranges of pre-RFCA vs. post-RFCA were 1.03:0.60 and 0.49:0.28, respectively). Besides, the plasma miR-144-3p and miR-425-5p expression level were not significantly different between post-RFCA and the control group (p=0.108 and 0.628, respectively) (Figure 4D-F).
3.9 Effect of miR-144-3p and miR-425-5p on atrial fibroblast proliferation and fibrosis
Atrial fibroblast proliferation and fibrosis were the hallmark of atrial remodeling in AF. We investigated whether the aberrant expression of the miRNAs influence fibroblast proliferation and fibrosis. The miR-144-3p and miR-425-5p inhibitor was transfected into the atrial fibroblast cell to downregulate the expression of miR-144-3p and miR-425-5p (Figure 5A). We found downregulation of miR-144-3p and miR-425-5p promoted the atrial fibroblast proliferation (Figure 5B-C), and the proliferation marker expression of Ki67 and Cyclin D (Figure 5D). Besides, it also promoted the fibrosis marker expression of collagen I and collagen III (Figures 5E-F).
3.10 CREB1 was the common direct target of miR-144-3p and miR-425-5p
Two widely available mammalian target prediction programs (miRDB and TargetScan7.2 databases) were used to predict the target gene of the miRNAs. 645 and 97 targets of miR-144-3p and miR-425-5p respectively were indicated by miRDB and TargetScan7.2. 20 of them were among the common targets for miR-144-3p and miR-425-5p (Online Figure 4), including DIP2C, MAPK6, CPEB2, ZNF148, ATP1B1, CREB1, LCOR, SYNCRIP, CPEB1, IFFO2, SCAMP1, TM9SF3, AFF4, FST, ZFHX3, CREBZF, LARP4B, FBN1, GOLGA4, SMAD5. We then after analyzed the expression level of 20 genes in the atrial tissue in SR and AF (Primer showed in the Online Table4). We found only the expression of CREB1 was increased. Besides, FBN1 and ZFHX3 expression was decreased in AF than in SR (Figure 6A). The expression of miRNA was negatively correlated with the target gene expression, on this basis, we speculated that CREB1 may be the target gene for miR-144-3p and miR-425-5p. More importantly, our results also showed the expression of miR-144-3p and miR-425-5p was negatively associated with CREB1 expression in the atrial tissue in AF (Figures 6B-C). Finally, we found that inhibition of CREB1 expression could reverse the phenotype of down-regulation of miR-144-3p and miR-425-5p(Figure 6D-F).
The potential binding target of miR-144-3p and miR-425-5p with CREB1 3’-URT was predicted by TargetScan 7.2 (Figure 7A), and it was realized that the downregulation of miR-144-3p and miR-425-5p significantly promoted CREB1 protein expression in the fibroblast cells (Figure 7B-C). Furthermore, through luciferase assay, CREB1 was verified as the common direct target for miR-144-3p and miR-425-5p (Figure7D-E).