Potential function and molecular mechanism of circRNAs involved in idiopathic pulmonary fibrosis

Background: Recent studies have found a regulatory role of circular RNAs (circRNAs) in the pathogenesis of idiopathic pulmonary fibrosis (IPF). However, the function and underlying molecular mechanism of circRNAs involved in IPF are uncertain and incomplete. This study aimed to further provide some critical information for the circRNA function in IPF using bioinformatic analysis. Methods: We searched in the NCBI (National Center for Biotechnology Information) Gene Expression Omnibus (GEO) database to find the circRNA expression profiles of human IPF. The microarray data GSE102660 was obtained and differentially expressed circRNAs were identified through R software. Results: 6 significantly up-regulated and 13 significantly down-regulated circRNAs were identified involved in the pathogenesis of IPF. The binding sites of miRNAs for each differentially expressed circRNA were also predicted and circRNA-miRNA-mRNA networks were constructed for the most up-regulated hsa_circ_0004099 and down-regulated hsa_circ_0029633. In addition, GO and KEGG enrichment analysis revealed the molecular function and enriched pathways of the target genes of circRNAs in IPF. Conclusion: These findings suggest that candidate circRNAs might serve an important role in the pathogenesis of IPF. Therefore, these circRNAs might be potential biomarkers for diagnosis and promising targets for treatment of IPF, which still need further veriﬁcation in vivo and in vitro.


Background
Idiopathic pulmonary fibrosis (IPF) is a specific form of chronic, progressive, and irreversible interstitial lung disease (ILD) of unknown cause [1], regarded as a consequence of multiple interacting genetic and environmental risk factors, which leads to myofibroblast activation and consequent abnormal accumulation of extracellular matrix [2]. Patients with IPF generally suffer progressive dyspnea, a decline in lung function, decreased activity tolerance and poor quality of life [3][4][5]. Since current antifibrotic treatment can only delay disease progression to some extent but does not cure the disease itself, the prognosis of IPF is poor, with a 5-year survival rate between 20% and 40%, which is lower than many tumors [6][7][8]. Therefore, it is important to explore the molecular mechanisms responsible for IPF, aiming to find new methods to prevent and treat IPF. Circular RNAs (circRNAs) are a group of noncoding RNAs (ncRNAs) with a closed continuous loop structure, lacking terminal 5′ caps and 3′ tails [9]. They are generated in the process of back-splicing and often dynamically expressed among various tissues and cell types [10,11]. Generally, circRNAs regulate gene expression via different modes [12]: (a) Act as miRNA sponges by competing for miRNA binding sites; (b) interact with RNAbinding proteins (RBPs); (c) sequester mRNA translation start sites; (d) encode proteins.
Recently, an increasing number of studies have found a regulatory function of circRNAs in several diseases, including cancers, cardiovascular diseases, neurological disorders, diabetes and infection [13][14][15][16][17]. Li et al have found some dysregulated circRNAs in IPF through microarrays [18]. However, the function and underlying molecular mechanism of circRNAs involved in IPF are uncertain and incomplete. In the present study, comprehensive bioinformatic analysis were conducted to further provide some critical information for the function of circRNAs in IPF.

Methods
Identify differentially expressed circRNA profiles in IPF We searched in the NCBI (National Center for Biotechnology Information) Gene Expression Omnibus (GEO) database [19] to find the circRNA expression profiles of human IPF. The microarray data GSE102660 including 3 plasma samples from IPF patients and 3 plasma samples from healthy individuals was obtained and differentially expressed circRNAs were identified through R software (version 3.6.1). Limma package was used to find the different expression between control group and IPF group. P-value < 0.05 and |log 2 (fold change) | > 1 were considered to be significantly different. Subsequently, a volcano plot and a heat map were used to display the differential expression of circRNAs between the two groups.
Verify circRNA targeting miRNAs databases. To make our results more convincible, only the target genes predicted by all three databases were selected for further analysis. Finally, the circRNA-miRNA-mRNA interaction network was drawn using Cytoscape software (version 3.7.1).

Analyze Molecular Function And Enriched Pathways For circRNA-targeting
Genes Molecular function of the circRNA-target genes was further analyzed using GO enrichment.
In addition, enriched pathways of circRNA-target genes were identified using KEGG analysis. The GO and KEGG analysis were performed using R software. The P-value < 0.05 was considered significant. A bar and a bubble chart were used to present the molecular function and enriched pathways for circRNAtargeting genes.

Results
Identification of differentially expressed circRNA profiles in IPF Based on the analysis of GSE102660, differentially expressed circRNAs were identified using R language. It was considered significantly different that P-value < 0.05 and |log 2 (fold change) | > 1 compared to control group. As shown in Fig. 1A, a volcano plot visualized the differential expression of circRNAs between the IPF group and control group. Figure 1B revealed the differential expression profiles of circRNAs in the two groups using hierarchical cluster analysis. Finally, 19 differentially expressed circRNAs were identified between the IPF group and control group, of which 6 circRNAs were significantly up-regulated and 13 circRNAs were significantly down-regulated. The details are listed in Table 1.   Table 2. Of these, the top 5 potential miRNA binding sites for the most up-regulated circRNA (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 the most down-regulated circRNA (hsa_circ_0029633) were miR-124-3p, miR-223-5p, miR-3658, miR-486-5p as well as miR-630.

GO And KEGG Enrichment Analysis For circRNA-targeting Genes
The most up-regulated hsa_circ_0004099 and down-regulated hsa_circ_0029633 were selected for further investigation by Gene Ontology (GO; geneontology.org) and Kyoto Encyclopedia of Genes and Genomes (KEGG; www.kegg.jp) analysis. For hsa_circ_0004099, the target genes mainly participated in the molecular function of proximal promoter sequence-specific DNA binding, RNA polymerase II proximal promoter sequence-specific DNA binding and core promoter binding (P < 0.05), as shown in Fig. 4A and Table 3. KEGG pathways were also identified for the target genes of hsa_circ_0004099. Figure 4B and Table 4 presented that the enriched pathways included hepatitis B, measles, kaposi sarcoma-associated herpesvirus infection, p53 signaling pathway, thyroid hormone signaling pathway, cell cycle, and so on (P < 0.05). hsa_circ_0004099targeting genes that participated in cell cycle and p53 signaling pathway were shown in Additional file 1: S1 and Additional file 2: S2. For hsa_circ_0029633, the molecular function of target genes was mainly enriched in cell adhesion molecule binding, cadherin binding, molecular adaptor activity, protein binding, chromatin DNA binding, and so on, as shown in Fig. 5A and Table 5. Figure 5B and Table 6 showed that the enriched pathways for target genes of hsa_circ_0029633 involved non-small cell lung cancer, cellular senescence, focal adhesion, glioma, acute myeloid leukemia, PI3K-Akt signaling pathway, estrogen signaling pathway, prolactin signaling pathway, prolactin signaling pathway, and so on.
hsa_circ_0029633targeting genes that participated in ErbB, EGFR tyrosine kinase inhibitor resistance, FOXO, Ras, sphingolipid and PI3K-AKT signaling pathways were shown in Additional file 3: S3-Additional file 8: S8.      Discussion 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 noncoding RNA family and play an important role in the development of multiple lung diseases [26][27][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, 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][35][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  Therefore, these circRNAs might be potential biomarkers for diagnosis and promising targets for treatment of IPF, which still need further verification in vivo and in vitro.
Not applicable.

Funding
This work was supported by Chinese National Science Foundation (No. 81960014) and Cuiying Scientific and Technological Innovation Program of Lanzhou University Second Hospital (No. CY2018-QN12).

Supplementary Files
This is a list of supplementary files associated with the primary manuscript. Click to download. S7.tif