Identification of differentially expressed lncRNAs in AF patients
We obtained the circulating lncRNA profiling in AF patients and healthy controls from two cohorts. Both of the two cohorts consisted of blood samples from 3 AF patients and 3 healthy controls and the lncRNAs expression was profiled by using the Arraystar Human LncRNA Array. A total of 8,164 lncRNAs were annotated and analyzed in these two cohorts in this study. Based on the cut‐off criteria of P value < 0.01 and fold change > 2.0 or < 0.5, we identified 116 and 639 differentially expressed circulating lncRNAs in AF patients, respectively (Figure 1A and 1B). Among these differentially expressed lncRNAs, 49 and 251 were up‐regulated in these two cohorts, respectively (Figure 1C, top). Meanwhile, 67 and 388 lncRNAs were down-regulated in these two cohorts (Figure 1C, bottom). Two (LINC00174 and AP001360.3) and eight (AC109460.1, AC109460.3, AL031123.1, AL358113.1, MIAT, MIR4713HG, PTPRG-AS1 and ZNF815P) lncRNAs were significantly up-regulated and down-regulated in both the two cohorts, respectively (Figure 1C). Through unsupervised clustering analysis, the identified lncRNAs can categorize the blood samples into AF patients and healthy controls groups, showing good diagnostic prediction ability (Figure 1D and 1E).
Identification of differentially expressed mRNAs in AF patients
We then systematically searched for differentially expressed mRNAs in AF patients among 14,508 mRNAs in the two cohorts. Based on the same criterial as significant dysregulation lncRNA identification, we identified 143 and 869 differentially expressed circulating mRNAs in AF patients from the two cohorts, respectively (Figure 2A and 2B). Among these differentially expressed mRNAs, only one (TEAD2) was significantly up‐regulated in both the two cohorts with P value being 0.0078 (fold change = 2.23) and 0.0005 (fold change = 2.81) in GSE64904 and GSE75092, respectively (Figure 2C, top). Meanwhile, six mRNAs (D2HGDH, KCND2, PTPRG, SOCS3, SPNS1 and TNFAIP8L3) were significantly down-regulated in both the two cohorts (Figure 2C, bottom). Similarly, these mRNAs also showed great diagnostic prediction potential that can categorize the blood samples into AF patients and healthy controls by using unsupervised clustering analysis (Figure 2D and 2E).
Identification of lncRNA-mRNA network
To further investigate the potential function of these identified dysregulated lncRNAs and mRNAs in AF patients, we conducted co-expression analysis by using the RNA-seq data of 429 normal Atrial Appendage tissue samples from the Genotype-Tissue Expression (GTEx) project database. Significant correlation (Pearson P value < 0.05 and r > 0.3 or <-0.3) between the differentially expressed lncRNAs and mRNAs were shown in the Table 1. The most significant co-expression was PTPRG-AS1 and D2HGDH with Pearson P value being 3.69×10-36 and correlation r being 0.56. By using these significant lncRNA and mRNA co-expression, we construct a potential lncRNA-mRNA network (Figure 3A). As all the significant lncRNA-mRNA co-expressions were positive correlation (Table 1), we hypotheses a miRNA sponge mechanism for these lncRNAs. Therefore, we performed miRNA target prediction for these lncRNAs and mRNAs by using a transcriptome-wide miRNA target prediction tool including lncRNAs (miRcode) [18], which was based on Targetscan 6. The results showed that the lncRNA MIAT may regulate SPNS1 and D2HGDH by competitive binding of miR-15, miR-24, miR-27 et al (Figure 3B, left) and the PTPRG-AS1 also serve as a miRNA sponge to regulate D2HGDH, SPNS1, TNFAIP8L3 and KCND2 (Figure 3B, right). Collectively, these results indicate a potential dysregulated lncRNA-miRNA-mRNA network may function in both the blood and the Arial tissues of AF patients.
Table 1
Significant co-expression of identified dysregulation lncRNAs and mRNAs in AF
LncRNAs
|
LncRNA id
|
mRNAs
|
Pearson P
|
r
|
AC109460.1
|
ENSG00000260367.2
|
SPNS1
|
2.00×10-26
|
0.48
|
|
|
D2HGDH
|
1.54×10-14
|
0.36
|
AL031123.1
|
ENSG00000226281.2
|
SPNS1
|
6.01×10-12
|
0.32
|
|
|
D2HGDH
|
2.95×10-11
|
0.31
|
MIAT
|
ENSG00000225783.6
|
SPNS1
|
2.00×10-26
|
0.48
|
|
|
D2HGDH
|
1.54×10-14
|
0.36
|
PTPRG-AS1
|
ENSG00000241472.6
|
D2HGDH
|
3.69×10-36
|
0.56
|
|
|
SPNS1
|
6.25×10-22
|
0.44
|
|
|
TNFAIP8L3
|
1.84×10-11
|
0.32
|
|
|
KCND2
|
1.27×10-10
|
0.30
|
ZNF815P
|
ENSG00000235944.7
|
SPNS1
|
1.17×10-39
|
0.58
|
|
|
D2HGDH
|
2.92×10-37
|
0.56
|