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Identification and Validation of Genes Exhibiting Dynamic Alterations in Response to Bleomycin-Induced Pulmonary Fibrosis

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Abstract

Idiopathic pulmonary fibrosis (IPF) carries a high mortality rate and has a poor prognosis. The pathogenesis of pulmonary fibrosis (PF) is highly related to dysregulation of multiple RNAs. This study aims to identify and validate dysregulated RNAs that exhibited dynamic alterations in response to bleomycin (BLM)-induced PF. The results will provide therapeutic targets for patients suffering from IPF. Whole transcriptomic profiles of BLM-induced PF were obtained through high-throughput RNA sequencing. miRNA profiling was downloaded from GSE45789 database in the Gene Expression Omnibus (GEO). We identified the differentially expressed RNAs (DERNAs) that exhibited dynamic alterations in response to BLM-induced PF. Subsequently, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genome (KEGG) pathway enrichment analysis were conducted to discovery regulatory processes of PF. Weighted gene co-expression network analysis (WGCNA), protein–protein interaction (PPI) analysis, and co-expression analysis were performed to identify key genes and pathogenic pattern during the progression of PF. MiRanda, miRcode, and TargetScan were utilized to predict target relationships in the potential competing endogenous RNA (ceRNA) network. The results were verified by qRT-PCR analysis. In the context of BLM-induced PF, this study identified a total of 167 differentially expressed messenger RNAs (DEmRNAs), 115 differentially expressed long non-coding RNAs (DElncRNAs), 45 differentially expressed circular RNAs (DEcircRNAs), and 87 differentially expressed microRNAs (DEmiRNAs). These RNA molecules showed dynamic alterations in response to BLM-induced PF. These DEmRNAs exhibited a predominant association with the biological processes pertaining to the organization of extracellular matrix. A regulatory network was built in PF, encompassing 31 DEmRNAs, 18 DE lncRNAs, 13 DEcircRNAs, and 13 DEmiRNAs. Several DERNA molecules were subjected to validate using additional BLM-induced PF model. The outcomes of this validation process shown a strong correlation with the results obtained from RNA sequencing analysis. The GSE213001 dataset was utilized to validate the expression levels and diagnostic efficacy of four specific hub mRNAs (CCDC80, CLU, COL5A1, and COL6A3) in individuals diagnosed with PF. In this study, we identified and validated several RNA molecules that exhibited dynamic alternations in response to BLM-induced PF. These dysregulated RNAs participated in the pathogenesis of PF and can be used as therapeutic targets for early-stage IPF. Although more work must be done to confirm the results, our study may provide directions for future studies.

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Data Availability

NGS data are available from the NCBI Sequence Read Archive database (BioProject: PRJNA962191).

Abbreviations

IPF:

Idiopathic pulmonary fibrosis

ILD:

Interstitial lung disease

GEO:

Gene Expression Omnibus

DERNAs:

Differentially expressed RNAs

CeRNA:

Competiting endogenous RNA

GO:

Gene Ontology

KEGG:

Kyoto Encyclopedia of Genes and Genome

PPI:

Protein–protein interaction

WGCNA:

Weighted gene co-expression network analysis

ncRNAs:

Non-coding RNAs

PCA:

Principal component analysis

FMT:

Fibroblast-to-myofibroblast transition

EMT:

Epithelial-mesenchymal transition

BLM:

Bleomycin

qRT-PCR:

Quantitative Real-Time PCR

DEGs:

Differentially expressed genes

ROC:

Receive operating characteristic

AUCs:

The area under the curve

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Acknowledgements

Not applicable.

Funding

This work was supported by The National Natural Science Foundation of China (89202586), Joint Application Foundation Project of Kunming Medical University and Yunnan Provincial Science and Technology Department (202101AY070001-271 and 202001AY070001-289), and Open Project of Yunnan Provincial Clinical Medical Center for Respiratory System Diseases (2020LCZXKF-HX08, 2022LCZXKF-HX04).

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Author notes

  1. Dengyuan Li and Jun Wang have contributed equally to this work and share first authorship.

Authors and Affiliations

  1. Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming, 650500, China

    Dengyuan Li

  2. Department of Pulmonary and Critical Care Medicine, The First People’s Hospital of Yunnan Province, The Affiliated Hospital of Kunming University of Science and Technology, Kunming, 650022, China

    Dengyuan Li, Jie Zeng, Danxiong Sun, Zhenming Huang & Yunhui Zhang

  3. Shenzhen Key Laboratory of Microbial Genetic Engineering, Vascular Disease Research Center, College of Life Sciences and Oceanography, Guangdong Provincial Key Laboratory of Regional Immunity and Disease, Carson International Cancer Center, School of Medicine, Shenzhen University, Shenzhen, 518060, China

    Jun Wang, Shujin Li, Lin Qiu, Ku Wang, Gaohui Fu & Deming Gou

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Contributions

YHZ and DMG: designed the study. DYL and JZ: constructed the BLM-induced PF mice model. JW, SJL, LQ, GHF and KW: performed comprehensive bioinformatics analysis. DYL, DXS, and ZMH: performed the qRT-PCR validation. DYL: wrote the article. JW, DMG and YHZ: revised the article. All authors approved the final version of the manuscript.

Corresponding authors

Correspondence to Deming Gou or Yunhui Zhang.

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The authors declare that they have no competing interests.

Ethical approval

All procedures comply with National Institutes of Health guidelines (NIH Publication, 8th edition, 2011), and were approved by the Institutional Animal Care and Use Committee (IACUC) of The First People’s Hospital of Yunnan Province.

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Li, D., Wang, J., Zeng, J. et al. Identification and Validation of Genes Exhibiting Dynamic Alterations in Response to Bleomycin-Induced Pulmonary Fibrosis. Mol Biotechnol (2023). https://doi.org/10.1007/s12033-023-00943-4

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