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Construction of potential idiopathic pulmonary fibrosis related microRNA and messenger RNA regulatory network

To the Editor: Idiopathic pulmonary fibrosis (IPF) is a kind of lung disease characterized by chronic and progressive pulmonary fibrosis with unknown etiology, whose pathological manifestation is usual interstitial pneumonia. However, the prognosis of IPF was still poor, and the median survival time after diagnosis was about 2 to 3 years. Therefore, it seems important to seek and develop an effective therapeutic modality for IPF. Through base pairs of intramolecular complementary sequences of messenger RNA (mRNA), microRNA (miRNA) plays a key role in RNA silencing and post-transcriptional gene expression regulation. Each miRNA can have multiple target genes and several miRNAs can co-regulate one gene. They also play an important role in organfibrosis, especially in IPF, and the rolemay be related to the pathogenesis of the disease. Construction of potential miRNA-mRNA regulatory network of IPF will help to reveal more comprehensive molecular mechanism of miRNAs in IPF.

To the Editor: Idiopathic pulmonary fibrosis (IPF) is a kind of lung disease characterized by chronic and progressive pulmonary fibrosis with unknown etiology, whose pathological manifestation is usual interstitial pneumonia. [1] However, the prognosis of IPF was still poor, and the median survival time after diagnosis was about 2 to 3 years. [2] Therefore, it seems important to seek and develop an effective therapeutic modality for IPF. Through base pairs of intramolecular complementary sequences of messenger RNA (mRNA), microRNA (miRNA) plays a key role in RNA silencing and post-transcriptional gene expression regulation. [3] Each miRNA can have multiple target genes and several miRNAs can co-regulate one gene. They also play an important role in organ fibrosis, especially in IPF, and the role may be related to the pathogenesis of the disease. [4,5] Construction of potential miRNA-mRNA regulatory network of IPF will help to reveal more comprehensive molecular mechanism of miRNAs in IPF. We used Enrichr database, a comprehensive gene set enrichment analysis web server (http://amp.pharm.mssm. edu/Enrichr/) to perform gene ontology (GO) function enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of the screened candidate target genes of DE-miRNAs.
GO biological process (BP) analysis showed that downregulated candidate target genes (candidate target genes of up-regulated DE-miRNAs) were significantly enriched in negative regulation of DNA binding, regulation of adiponectin secretion, positive regulation of cell migration by vascular endothelial growth factor signaling pathway, and so on. GO molecular function (MF) analysis showed that candidate target genes of up-regulated DE-miRNAs were significantly enriched in transcription corepressor binding, G-protein coupled receptor activity, postsynaptic density protein-95, Drosophila disc large tumor suppressor, and zonula occludens-1 (PDZ) domain binding, and so on. GO cellular component (CC) analysis showed that candidate target genes of up-regulated DE-miRNAs were significantly enriched in integral component of plasma membrane, clathrin-coated endocytic vesicle membrane, clathrin-coated endocytic vesicle, and so on.
GO BP analysis showed that up-regulated candidate target genes (candidate target genes of down-regulated DE-miRNAs) were significantly enriched in positive regulation of gene expression, positive regulation of nucleic acid-templated transcription, negative regulation of neuron apoptotic process, and so on. GO MF analysis showed that candidate target genes of down-regulated DE-miRNAs were significantly enriched in metalloendopeptidase activity, TF activity/RNA polymerase II core promoter proximal region sequence-specific binding, transcriptional activator activity/RNA polymerase II core promoter proximal region sequence-specific binding, and so on. GO CC analysis showed that candidate target genes of down-regulated DE-miRNAs were significantly enriched in platelet alpha granule membrane, platelet alpha granule, dendrite, and so on.
KEGG pathway enrichment analysis was further conducted for candidate target genes of DE-miRNAs. Candidate target genes of up-regulated DE-miRNAs (down-regulated candidate target genes) were significantly enriched in pathways in cancer, signaling pathways regulating pluripotency of stem cells, breast cancer, and so on. Candidate target genes of down-regulated DE-miRNAs (up-regulated candidate target genes) were significantly enriched in bladder cancer, central carbon metabolism in cancer, transcriptional misregulation in cancer, and so on.
We mapped these candidate target genes into the STRING database (https://string-db.org/). STRING is one of ELIXIR's Core Data and a web server to retrieve and display the repeatedly occurring neighborhood of a gene. We found out that 44 candidate target genes of upregulated DE-miRNAs in the network could have protein interaction and 116 edges represented the interaction between proteins, while 51 candidate target genes of down-regulated DE-miRNAs in the network could have protein interaction and 209 edges represented the interaction between proteins.
Then we input these nodes and edges data into Cytoscape 3.6.0 software and used the "Network Analyzer" tools to do topology analysis. Then the hub genes were identified. The top 12 hub genes in the target genes of up-regulated DE-miRNAs were VEGFA, FOS, IFNG, MAPK4, MAP2, FABP4, PTGDR, GRM3, KLF6, SPRY4, PER2, and HEY1; the top 12 hub genes in the target genes of downregulated DE-miRNAs were MMP9, SOX2, MMP2, CDH2, TOP2A, HIF1A, MMP1, IGFBP5, CALU, HMGA2, ITGB3, and CLU. According to the miRNA and candidate target gene pairs analyzed above, we found a link between miRNA and hub genes [ Figure 1].
For our next study to be more targeted, GEO Dataset was still used to detect the 24 hub genes expression levels. We searched datasets focusing on the gene expression in GEO Dataset. Finally, GSE72073 was selected for subsequent analysis. The results showed that in GSE72073 dataset the expression level of VEGFA was significantly lower in IPF tissues than that in normal tissues and the expression levels of SOX2, MMP2, CDH2, TOP2A, HIF1A, MMP1, CALU, and ITGB3 were significantly higher in IPF tissues than those in normal tissues. Based on the preliminary validation, a more accurate potential miRNA-mRNA regulatory network contributing to IPF was established, including miR-410-3p/miR-495-3p-VEGFA, miR-375-SOX2/ CALU, miR-338-3p-MMP2/CDH2/HIF1A/ITGB3, and miR-203a-3p-TOP2A/MMP1 regulatory pathways, which could be further studied in clinical and basic experiments, combining the predicted TFs, GO enrichment analysis and KEGG pathways analysis above.
In conclusion, we reveals a comprehensive potential mechanisms of miRNA-mRNA regulatory axes in pathogenesis of IPF and established a potential IPF related miRNA-mRNA regulatory network, which may help to know the underlying mechanism and new research pathway of IPF.