IPF is a progressive and ultimately fatal interstitial lung disease, whose available therapies are limited [2, 25]. Deeply to explore the pathogenesis and further to find new therapeutic options for IPF are a clear and urgent need. circRNAs are a class of the non-coding RNA family and play an important role in the development of multiple lung diseases [26–28]. Recently, studies have detect circRNAs with abnormal expression in IPF using a high‑throughput microarray assay and found that several circRNAs may be potential biomarkers and promising molecular targets for the diagnosis and treatment of IPF [18, 29]. However, the function and underlying molecular mechanism of circRNAs contributing to the development of IPF remain largely uncertain and incomplete.
In this study, 6 significantly up-regulated and 13 significantly down-regulated circRNAs were identified based on the analysis of GSE102660 through R software. Of these, the most up-regulated circRNA (hsa_circ_0004099) and down-regulated circRNA (hsa_circ_0029633) were chosen for further analysis. The top 5 potential miRNA binding sites for hsa_circ_0004099 were miR-4633-5p, miR-3671, miR-4755-3p, miR-665 as well as miR-9-3p, and the top 5 potential miRNA binding sites for hsa_circ_0029633 were miR-124-3p, miR-223-5p, miR-3658, miR-486-5p as well as miR-630. Next, target genes of the top 5 miRNAs for hsa_circ_0004099 and hsa_circ_0029633 were predicted and the circRNA-miRNA-mRNA interaction networks were constructed. Finally, GO analysis showed that target genes of hsa_circ_0004099 were enriched in proximal promoter sequence-specific DNA binding, RNA polymerase II proximal promoter sequence-specific DNA binding as well as core promoter binding, and target genes of hsa_circ_0029633 were enriched in many biological processes, such as cell adhesion molecule binding, molecular adaptor activity, chromatin DNA binding, signaling adaptor activity as well as phosphoprotein phosphatase activity, and so on. In KEGG pathway analysis, the target genes of hsa_circ_0004099 were mainly located in p53 signaling pathway, thyroid hormone signaling pathway, Epstein-Barr virus infection and cell cycle signaling pathways; while the target genes of hsa_circ_0029633 were mainly located in focal adhesion signaling pathway, PI3K-Akt signaling pathway, FOXO signaling pathway, EGFR tyrosine kinase inhibitor resistance signaling pathway, Hippo signaling pathway, ErbB signaling pathway as well as Ras signaling pathway, and so on. These results indicated that hsa_circ_0004099 and hsa_circ_0029633 might regulate crucial biological processes during the development of IPF.
Several studies have shown that dysregulation of miR-4633 and miR-9
is involved in the pathogenesis of IPF [30, 31], and increased expression of IGFBP5 and ITGB1 plays a vital role in the development of IPF [32, 33]. Our present results suggested that up-regulated hsa_circ_0004099 might promote the development of IPF by decreasing the expression of miR-4633 or miR-9, and further increasing the expression of IGFBP5 or ITGB1, respectively. MAP3K7, TBK1 and ETS2 have been proved crucial factors in IPF progression [34–36], and these genes were predicted as downstream targets of miR-3671, which was negatively modulated by hsa_circ_0004099 in our analysis, suggesting that over-expression of hsa_circ_0004099 induced pulmonary fibrosis via reduction of miR-3671 and consequent increase of MAP3K7, TBK1 or ETS2.
In our analysis, many genes such as SMAD5, CAV1, JAG1, ROCK1 and STAT3 were positively predicted as downstream targets of miR-124, which was regarded as a potential miRNA binding site of hsa_circ_0029633. Previous studies have demonstrated that miR-124 plays a key role in multiple diseases including IPF by targeting SMAD5, CAV1, JAG1, ROCK1 or STAT3 [37–42], which is consistent with our findings. Previous studies have also shown that FOXC1 and HSPA1A are direct target genes of miR-223 [43, 44], and these genes contribute to the pathogenesis of IPF via activating various signaling pathways. Our present results suggested that down-regulated hsa_circ_0029633 might promote the development of IPF by increasing the expression of miR-223, and further decreasing the expression of FOXC1 and HSPA1A. In addition, we found that hsa_circ_0029633/ miR-486-5p and hsa_circ_0029633/ miR-630 signaling axes were also involved in the development of IPF via targeting CADM1 and OLFM4, as well as PDGFRA and YAP1, respectively.
Several published studies have demonstrated that up-regulation of miR-486-5p and miR-630 contributes to IPF progression [45, 46] and CADM1, OLFM4, PDGFRA as well as YAP1 genes play a crucial role in the pathogenesis of IPF [47–50].
Some limitations should be addressed when interpreting the results: (a) The differentially expressed circRNAs were identified only based on microarray data GSE102660 and they still needed further verification in animal and human lungs; (b)
the function of the differentially expressed circRNAs in IPF was predicted only using bioinformatic analysis, and there was no further study in vivo or in vitro to demonstrate the roles of candidate circRNAs in the pathogenesis of IPF; (c) generally, circRNAs regulate gene expression via several modes , such as acting as miRNA sponges, interacting with RBPs, sequestering mRNA translation start sites and encoding proteins, while the current study was performed only according to the miRNA sponge function of circRNAs.