Identification of significantly different expressed miRNAs (DEMs)
We screened out DEMs in each time period (Fig. 1A、B、C). A total of 11 DEMs were obtained, with 5 up-regulated and 6 down-regulated, in the sham operation group and 1.5 days after surgery. Studies have shown that the expression of miRNA-21-3p is up-regulated, and there is no significant difference in the expression of miRNA-21-5p. A total of 16 DEMs were obtained, with 7 up-regulated and 9 down-regulated, in the sham operation group and 3 days after surgery. And the expression of miRNA-21-3p was still up-regulated, and the difference of miRNA-21-5p was still not significant. On the 14th day after surgery, 9 miRNAs with up-regulated and 1 miRNA with down-regulated were screened, among which the expression of miRNA-21-3p was not significantly different, while the expression of miRNA-21-5p showed an up-regulated trend. The expression was completely contrary to that on 1.5 days and 3 days. Therefore, we believed that 3 days was the time node for AKI repair, and miRNA-21-3p and miRNA-21-5p had a mutual restriction relationship. Subsequently, the different genes screened in the three time periods were unionized, and a total of 29 miRNAs were obtained. Figure 1D more intuitively shows the expression changes of miRNA-21-3p, miRNA-21-5p and other significantly different miRNA over time. The original data was stored in Supplementary Material S1.
Target gene prediction and construction of regulatory networks
The target genes of 29 DEMs were predicted, and the PPI network of target genes were obtained (Supplementary Material S2). Then, the interactions between miRNAs-mRNA and mRNA- mRNA were visualized (Fig. 2). Studies have shown that the target genes of miRNA-21-3p include Cadm1、Fshb and Pten, and miRNA-21-5p include 19 target genes, such as Elavl4、Faslg and Pten. At the same time, we found that miRNA-21-3p and miRNA-21-5p jointly targeted the coding gene Pten. Thus, our study speculated that the expression of miRNA-21-3p and miRNA-21-5p was mutually restricted in the occurrence of AKI, so Pten was not differentially expressed in the competition between the two miRNAs.
Identification of significantly different expressed target genes (DEGs)
We performed a differential expression analysis to acquire DEGs between the group of 3 days after surgery and control, and the group of 3 days and 7 days after surgery (Supplementary Material S3). Then, 8 mRNAs which are the intersections of DEGs and target genes of miRNA-21-3p and miRNA-21-5p were obtained (Fig. 3 and Fig. 4). We can find that the expression of Pten is not significantly difference between two groups, which further confirms our speculation above. Then, based on the comparison between 3 days after surgery and control group, the expression of Cadm1 which is the target gene of miRNA-21-3p was up-regulated and Fshb was down-regulated, as well as the expression of Tagln, Pdpn and Tpm1 which are target genes of miRNA-21-5p were up-regulated, Timp3 and Kcnj16 were down-regulated, and Nqo1 was no significantly difference. The comparison analysis on the 7th and 3rd day after surgery showed that the expression of target genes of miRNA-21-3p, including Cadm1 and Fshb, had no significant difference, and the expression of target genes of miRNA-21-5p, including Pdpn, Timp3 and Kcnj16, had no significant difference, as well as Tagln、Nqo1 and Tpm1 were down-regulated. Therefore, based on the change of time period, our study believe that miRNA-21-3p negatively regulated the expression of Fshb, and miRNA-21-5p negatively regulated the expression of target genes which are Tagln, Pdpn and Tpm1 to influence the occurrence and development of AKI and the progression of AKI to CKD.
The regulatory relationship of TFs-miRNA-mRNA
Based on TransmiR v2.0 software, we obtain three TFs, associated with kidney disease, which can regulate the expression of pri-mir-21(Table 1), including Hnf4a、Nfkb1、Rela. Interestingly, Rela is also a target gene of miRNA-21-5p, which does not meet the screening conditions. Additionally, in order to acquire the TFs of target genes which include Fshb, Tagln, Pdpn and Tpm1. The starting position, ending position (Table 2) and sequence of the promoter of target genes were obtained (Supplementary Material S4). Then, the promoter sequences and the vertebrate MEME files are uploaded to the MEME Suite's subdatabase-FIMO, and 1573 motifs which include TFs were obtained (Supplementary Material S5). Subsequently, four items which were consistent with TFs of pri-mir-21 were screened out (Table 3). Finally, the schematic diagram of regulatory relationship among TFs-miRNA-mRNA was visualized (Fig. 5). Our study showed that TFs, including Hnf4a and Rela, regulate pri-miR-21, and pri-mir-21 formed mature miRNA-21-3p and miRNA-21-5p after splicing. Therefore, we believed that Hnf4a and Rela indirectly regulate the expression of miRNA-21-3p and miRNA-21-5p. In addition, Hnf4a can regulate the expression of Fshb and Tpm1, Rela can positively regulate the expression of Tagln and Tpm1. Therefore, the mutual regulation of TFs-miRNA-mRNA is a potential mechanism to study AKI.
Table 1
TFs regulating pri-mir-21
TFs | TSS | Binding site | Action type | Tissue |
Hnf4a | chr10: 73902301 | chr10: 73905311–73905440(score = 603) | Regulation | Kidney |
Hnf4a | chr10: 73902301 | chr10: 73905319–73905412(score = 393) | Regulation | Kidney |
Hnf4a | chr10: 73902301 | chr10: 73905351–73905445(score = 328) | Regulation | Kidney |
Nfkb1 | n/a | n/a | Activation | n/a |
Rela | n/a | n/a | Activation(feedback) | n/a |
*TSS: Transcription start site. |
Table 2
Promoter sequence information of Fshb, Tagln, Pdpn and Tpm1
Gene name | Chr start | Chr end | Promoter start | Promoter end |
Fshb | 93548560 | 93552370 | 93546560 | 93548559 |
Tagln | 46224939 | 46230413 | 46222939 | 46224938 |
Pdpn | 155601691 | 155635656 | 155599691 | 155601690 |
Tpm1 | 67635479 | 67662330 | 67633479 | 67635478 |
Table 3
The TFs of regulating Fshb、Tagln、Pdpn and Tpm1
Motif | TF | Gene | Start | Stop | Strand | Score | p-value | Matched Sequence |
MA0114.4 | Hnf4a | Tpm1 | 252 | 264 | - | 11.6525 | 4.79E-05 | GGAAAAGTCCACA |
MA0114.4 | Hnf4a | Fshb | 994 | 1006 | - | 11.2057 | 6.45E-05 | CCCAAAGACCAGG |
MA0107.1 | Rela | Tpm1 | 1463 | 1472 | - | 9.73469 | 7.07E-05 | CTGGCTTTCC |
MA0107.1 | Rela | Tagln | 1480 | 1489 | - | 9.04082 | 8.27E-05 | TTGGCTTTCC |
Functional enrichment analysis of target genes
Functional enrichment analysis was performed for four target genes and revealed multiple critical GO Terms functions and KEGG pathway (Fig. 6, Supplementary Material S6). The results showed that target genes are enriched in some biological processes, including muscle organ development, positive regulation of cell adhesion, lymphangiogenesis, epithelial cell fate commitment, regulation of cell morphogenesis, peptide hormone processing, etc. Cellular components enriched by target genes is actin filament. Some molecular functions enriched by target genes include actin binding, chemokine binding, chaperone binding. The KEGG pathway were enriched to Ovarian steroidogenesis, Cardiac muscle contraction, Hypertrophic cardiomyopathy, GnRH signaling pathway and Dilated cardiomyopathy. Nevertheless, the mechanisms which target genes regulate the progression of AKI to CKD need to be confirmed by further studies.