Integrated whole transcriptome analysis for the crucial regulators and functional pathways related to cardiac fibrosis in rats

Shuai Miao; Lijun Wang; Siyu Guan; Tianshu Gu; Hualing Wang; Wenfeng Shangguan; Weiding Wang; Yu Liu; Xue Liang; Shuai Miao; Lijun Wang; Siyu Guan; Tianshu Gu; Hualing Wang; Wenfeng Shangguan; Weiding Wang; Yu Liu; Xue Liang

doi:10.3934/mbe.2023250

Mathematical Biosciences and Engineering

2023, Volume 20, Issue 3: 5413-5429. doi: 10.3934/mbe.2023250

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Research article

Integrated whole transcriptome analysis for the crucial regulators and functional pathways related to cardiac fibrosis in rats

1.
Tianjin Key Laboratory of Ionic-Molecular Function of Cardiovascular disease, Department of Cardiology, Tianjin Institute of Cardiology, the Second Hospital of Tianjin Medical University, Tianjin 300211, China
2.
Department of Emergency Medicine, Tianjin Medical University General Hospital, Tianjin 300052, China
3.
Taikang Ningbo Hospital, Ningbo 315100, Zhejiang, China

† These authors contributed equally.
Academic Editor: Ulf Schmitz

Received: 30 October 2022 Revised: 17 December 2022 Accepted: 25 December 2022 Published: 12 January 2023

Background
Cardiac fibrosis has gradually gained significance in the field of cardiovascular disease; however, its specific pathogenesis remains unclear. This study aims to establish the regulatory networks based on whole-transcriptome RNA sequencing analyses and reveal the underlying mechanisms of cardiac fibrosis.
Methods
An experimental model of myocardial fibrosis was induced using the chronic intermittent hypoxia (CIH) method. Expression profiles of long non-coding RNA (lncRNA), microRNA (miRNA), and messenger RNA (mRNA) were acquired from right atrial tissue samples of rats. Differentially expressed RNAs (DERs) were identified, and functional enrichment analysis was performed. Moreover, a protein-protein interaction (PPI) network and competitive endogenous RNA (ceRNA) regulatory network that are related to cardiac fibrosis were constructed, and the relevant regulatory factors and functional pathways were identified. Finally, the crucial regulators were validated using qRT-PCR.
Results
DERs, including 268 lncRNAs, 20 miRNAs, and 436 mRNAs, were screened. Further, 18 relevant biological processes, such as "chromosome segregation, " and 6 KEGG signaling pathways, such as "cell cycle, " were significantly enriched. The regulatory relationship of miRNA–mRNA–KEGG pathways showed eight overlapping disease pathways, including "pathways in cancer." In addition, crucial regulatory factors, such as Arnt2, WNT2B, GNG7, LOC100909750, Cyp1a1, E2F1, BIRC5, and LPAR4, were identified and verified to be closely related to cardiac fibrosis.
Conclusion
This study identified the crucial regulators and related functional pathways in cardiac fibrosis by integrating the whole transcriptome analysis in rats, which might provide novel insights into the pathogenesis of cardiac fibrosis.
- cardiac fibrosis,
- whole-transcriptome RNA sequencing,
- functional pathways,
- competing endogenous RNA,
- regulatory network
Citation: Shuai Miao, Lijun Wang, Siyu Guan, Tianshu Gu, Hualing Wang, Wenfeng Shangguan, Weiding Wang, Yu Liu, Xue Liang. Integrated whole transcriptome analysis for the crucial regulators and functional pathways related to cardiac fibrosis in rats[J]. Mathematical Biosciences and Engineering, 2023, 20(3): 5413-5429. doi: 10.3934/mbe.2023250

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Abstract

Background

Cardiac fibrosis has gradually gained significance in the field of cardiovascular disease; however, its specific pathogenesis remains unclear. This study aims to establish the regulatory networks based on whole-transcriptome RNA sequencing analyses and reveal the underlying mechanisms of cardiac fibrosis.

Methods

An experimental model of myocardial fibrosis was induced using the chronic intermittent hypoxia (CIH) method. Expression profiles of long non-coding RNA (lncRNA), microRNA (miRNA), and messenger RNA (mRNA) were acquired from right atrial tissue samples of rats. Differentially expressed RNAs (DERs) were identified, and functional enrichment analysis was performed. Moreover, a protein-protein interaction (PPI) network and competitive endogenous RNA (ceRNA) regulatory network that are related to cardiac fibrosis were constructed, and the relevant regulatory factors and functional pathways were identified. Finally, the crucial regulators were validated using qRT-PCR.

Results

DERs, including 268 lncRNAs, 20 miRNAs, and 436 mRNAs, were screened. Further, 18 relevant biological processes, such as "chromosome segregation, " and 6 KEGG signaling pathways, such as "cell cycle, " were significantly enriched. The regulatory relationship of miRNA–mRNA–KEGG pathways showed eight overlapping disease pathways, including "pathways in cancer." In addition, crucial regulatory factors, such as Arnt2, WNT2B, GNG7, LOC100909750, Cyp1a1, E2F1, BIRC5, and LPAR4, were identified and verified to be closely related to cardiac fibrosis.

Conclusion

This study identified the crucial regulators and related functional pathways in cardiac fibrosis by integrating the whole transcriptome analysis in rats, which might provide novel insights into the pathogenesis of cardiac fibrosis.

References

[1]	N. G. Frangogiannis, Cardiac fibrosis, Cardiovasc. Res., 117 (2021), 1450–1488. https://doi.org/10.1093/cvr/cvaa324 doi: 10.1093/cvr/cvaa324
[2]	S. Hinderer, K. Schenke-Layland, Cardiac fibrosis—A short review of causes and therapeutic strategies, Adv. Drug Deliv. Rev., 146 (2019), 77–82. https://doi.org/10.1016/j.addr.2019.05.011 doi: 10.1016/j.addr.2019.05.011
[3]	Z. G. Ma, Y. P. Yuan, H. M. Wu, X. Zhang, Q. Z. Tang, Cardiac fibrosis: new insights into the pathogenesis, Int. J. Biol. Sci., 14 (2018), 1645–1657. https://doi.org/10.7150/ijbs.28103 doi: 10.7150/ijbs.28103
[4]	S. Nattel, Molecular and cellular mechanisms of atrial fibrosis in atrial fibrillation, JACC Clin. Electrophysiol., 3 (2017), 425–435. https://doi.org/10.1016/j.jacep.2017.03.002 doi: 10.1016/j.jacep.2017.03.002
[5]	M. Gyöngyösi, J. Winkler, I. Ramos, Q. T. Do, H. Firat, K. McDonald, et al., Myocardial fibrosis: biomedical research from bench to bedside, Eur. J. Heart Fail., 19 (2017), 177–191. https://doi.org/10.1002/ejhf.696 doi: 10.1002/ejhf.696
[6]	M. P. Czubryt, T. M. Hale, Cardiac fibrosis: Pathobiology and therapeutic targets, Cell. Signal., 85 (2021), 110066. https://doi.org/10.1016/j.cellsig.2021.110066 doi: 10.1016/j.cellsig.2021.110066
[7]	D. W. Thomson, M. E. Dinger, Endogenous microRNA sponges: evidence and controversy, Nat. Rev. Genet., 17 (2016), 272–283. https://doi.org/10.1038/nrg.2016.20 doi: 10.1038/nrg.2016.20
[8]	J. J. Chan, Y. Tay, Noncoding RNA: RNA regulatory networks in cancer, Int. J. Mol. Sci., 19 (2018), 1310. https://doi.org/10.3390/ijms19051310 doi: 10.3390/ijms19051310
[9]	J. Beermann, M. T. Piccoli, J. Viereck, T. Thum, Non-coding RNAs in development and disease: Background, mechanisms, and therapeutic approaches, Physiol. Rev., 96 (2016), 1297–1325. https://doi.org/10.1152/physrev.00041.2015 doi: 10.1152/physrev.00041.2015
[10]	X. Liu, Y. Tong, D. Xia, E. Peng, X. Yang, H. Liu, Circular RNAs in prostate cancer: Biogenesis, biological functions, and clinical significance, Mol. Ther. Nucleic Acids, 26 (2021), 1130–1147. https://doi.org/10.1016/j.omtn.2021.10.017 doi: 10.1016/j.omtn.2021.10.017
[11]	L. Zhang, C. Li, X. Su, Emerging impact of the long noncoding RNA MIR22HG on proliferation and apoptosis in multiple human cancers, J. Exp. Clin. Cancer Res., 39 (2020), 271. https://doi.org/10.1186/s13046-020-01784-8 doi: 10.1186/s13046-020-01784-8
[12]	Q. Y. Gao, H. F. Zhang, Z. T. Chen, Z. T. Chen, Y. W. Li, S. H. Wang, Construction and analysis of a ceRNA network in cardiac fibroblast during fibrosis based on in vivo and in vitro data, Front. Genet., 11 (2020), 503256. https://doi.org/10.3389/fgene.2020.503256 doi: 10.3389/fgene.2020.503256
[13]	S. Ghafouri-Fard, A. Abak, S. F. Talebi, H. Shoorei, W. Branicki, M. Taheri, et al., Role of miRNA and lncRNAs in organ fibrosis and aging, Biomed. Pharmacother., 143 (2021), 112132. https://doi.org/10.1016/j.biopha.2021.112132 doi: 10.1016/j.biopha.2021.112132
[14]	F. Wei, X. Zhang, X. Kuang, X. Gao, J. Wang, J. Fan, Integrated analysis of circRNA-miRNA-mRNA-mediated network and its potential function in atrial fibrillation, Front. Cardiovasc. Med., 9 (2022), 883205. https://doi.org/10.3389/fcvm.2022.883205 doi: 10.3389/fcvm.2022.883205
[15]	X. Gu, Y. N. Jiang, W. J. Wang, J. Zhang, D. S. Shang, C. B. Sun, et al., Comprehensive circRNA expression profile and construction of circRNA-related ceRNA network in cardiac fibrosis, Biomed. Pharmacother., 125 (2020), 109944. https://doi.org/10.1016/j.biopha.2020.109944 doi: 10.1016/j.biopha.2020.109944
[16]	H. Liang, Z. Pan, X. Zhao, L. Liu, J. Sun, X. Su, et al., LncRNA PFL contributes to cardiac fibrosis by acting as a competing endogenous RNA of let-7d, Theranostics, 8 (2018), 1180–1194. https://doi.org/10.7150/thno.20846 doi: 10.7150/thno.20846
[17]	Y. Liu, Y. Zhu, S. Liu, J. Liu, X. Li, NORAD lentivirus shRNA mitigates fibrosis and inflammatory responses in diabetic cardiomyopathy via the ceRNA network of NORAD/miR-125a-3p/Fyn, Inflamm. Res., 70 (2021), 1113–1127. https://doi.org/10.1007/s00011-021-01500-y doi: 10.1007/s00011-021-01500-y
[18]	Z. W. Huang, L. H. Tian, B. Yang, R. M. Guo, Long noncoding RNA H19 acts as a competing endogenous RNA to mediate CTGF expression by sponging miR-455 in cardiac fibrosis, DNA Cell Biol., 36 (2017), 759–766. https://doi.org/10.1089/dna.2017.3799 doi: 10.1089/dna.2017.3799
[19]	B. F. Zhang, H. Jiang, J. Chen, Q. Hu, S. Yang, X. P. Liu, et al., LncRNA H19 ameliorates myocardial infarction-induced myocardial injury and maladaptive cardiac remodelling by regulating KDM3A, J. Cell. Mol. Med., 24 (2020), 1099–1115. https://doi.org/10.1111/jcmm.14846 doi: 10.1111/jcmm.14846
[20]	H. Dai, N. Zhao, H. Liu, Y. Zheng, L. Zhao, LncRNA nuclear-enriched abundant transcript 1 regulates atrial fibrosis via the miR-320/NPAS2 axis in atrial fibrillation, Front. Pharmacol., 12 (2021), 647124. https://doi.org/10.3389/fphar.2021.647124 doi: 10.3389/fphar.2021.647124
[21]	S. Zhang, N. Wang, Q. Ma, F. Fan, X. Ma, LncRNA TUG1 acts as a competing endogenous RNA to mediate CTGF expression by sponging miR-133b in myocardial fibrosis after myocardial infarction, Cell Biol. Int., 45 (2021), 2534–2543. https://doi.org/10.1002/cbin.11707 doi: 10.1002/cbin.11707
[22]	Z. Ma, K. Zhang, Y. Wang, W. Wang, Y. Yang, X. Liang, et al., Doxycycline improves fibrosis-induced abnormalities in atrial conduction and vulnerability to atrial fibrillation in chronic intermittent hypoxia rats, Med. Sci. Monit., 26 (2020), e918883. https://doi.org/10.12659/MSM.918883 doi: 10.12659/MSM.918883
[23]	B. Zhao, W. Wang, Y. Liu, S. Guan, M. Wang, F. Song, et al., Establishment of a lncRNA-miRNA-mRNA network in a rat model of atrial fibrosis by whole transcriptome sequencing, J. Interv. Card. Electrophysiol., 63 (2022), 723–736. https://doi.org/10.1007/s10840-022-01120-4 doi: 10.1007/s10840-022-01120-4
[24]	M. E. Ritchie, B. Phipson, D. Wu, Y. Hu, C. W. Law, W. Shi, et al., limma powers differential expression analyses for RNA-sequencing and microarray studies, Nucleic Acids Res., 43 (2015), e47. https://doi.org/10.1093/nar/gkv007 doi: 10.1093/nar/gkv007
[25]	L. Wang, C. Cao, Q. Ma, Q. Zeng, H. Wang, Z. Cheng, et al., RNA-seq analyses of multiple meristems of soybean: novel and alternative transcripts, evolutionary and functional implications, BMC Plant Biol., 14 (2014), 169. https://doi.org/10.1186/1471-2229-14-169 doi: 10.1186/1471-2229-14-169
[26]	M. B. Eisen, P. T. Spellman, P. O. Brown, D. Botstein, Cluster analysis and display of genome-wide expression patterns, Proc. Natl. Acad. Sci. U. S. A., 95 (1998), 14863–14868. https://doi.org/10.1073/pnas.95.25.14863 doi: 10.1073/pnas.95.25.14863
[27]	D. W. Huang, B. T. Sherman, R. A. Lempicki, Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources, Nat. Protoc., 4 (2009), 44–57. https://doi.org/10.1038/nprot.2008.211 doi: 10.1038/nprot.2008.211
[28]	D. W. Huang, B. T. Sherman, R. A. Lempicki, Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists, Nucleic Acids Res., 37 (2009), 1–13. https://doi.org/10.1093/nar/gkn923 doi: 10.1093/nar/gkn923
[29]	D. Szklarczyk, A. L. Gable, K. C. Nastou, D. Lyon, R. Kirsch, S. Pyysalo, et al., The STRING database in 2021: customizable protein-protein networks, and functional characterization of user-uploaded gene/measurement sets, Nucleic Acids Res., 49 (2021), D605–D612. https://doi.org/10.1093/nar/gkaa1074 doi: 10.1093/nar/gkaa1074
[30]	P. Shannon, A. Markiel, O. Ozier, N. S. Balia, J. T. Wang, D. Ramage, et al., Cytoscape: a software environment for integrated models of biomolecular interaction networks, Genome Res., 13 (2003), 2498–2504. https://doi.org/10.1101/gr.1239303 doi: 10.1101/gr.1239303
[31]	D. Betel, A. Koppal, P. Agius, C. Sander, C. Leslie, Comprehensive modeling of microRNA targets predicts functional non-conserved and non-canonical sites, Genome Biol., 11 (2010), R90. https://doi.org/10.1186/gb-2010-11-8-r90 doi: 10.1186/gb-2010-11-8-r90
[32]	A. P. Davis, C. J. Grondin, R. J. Johnson, D. Sciaky, J. Wiegers, T. C. Wiegers, et al., Comparative toxicogenomics database (CTD): update 2021, Nucleic Acids Res., 49 (2021), D1138–D1143. https://doi.org/10.1093/nar/gkaa891 doi: 10.1093/nar/gkaa891
[33]	L. Yang, L. P. Li, H. C. Yi, DeepWalk based method to predict lncRNA-miRNA associations via lncRNA-miRNA-disease-protein-drug graph, BMC Bioinf., 22 (2022), 621. https://doi.org/10.1186/s12859-022-04579-0 doi: 10.1186/s12859-022-04579-0
[34]	G. J. Gaspard, J. MacLean, D. Rioux, K. B. S. Pasumarthi, A novel β-adrenergic response element regulates both basal and agonist-induced expression of cyclin-dependent kinase 1 gene in cardiac fibroblasts, Am. J. Physiol. Cell Physiol., 306 (2014), C540–C550. https://doi.org/10.1152/ajpcell.00206.2013 doi: 10.1152/ajpcell.00206.2013
[35]	S. Chen, Y. Jiang, J. Zhao, Y. Zhu, Y. Zhu, Z. Cao, et al., Cyclin dependent kinase 1 (CDK1) activates cardiac fibroblasts via directly phosphorylating paxillin at Ser244, Int. Heart J., 60 (2019), 374–383. https://doi.org/10.1536/ihj.18-073 doi: 10.1536/ihj.18-073
[36]	C. J. Leader, G. T. Wilkins, R. J. Walker, The effect of spironolactone on cardiac and renal fibrosis following myocardial infarction in established hypertension in the transgenic Cyp1a1Ren2 rat, PloS One, 16 (2021), e0260554. https://doi.org/10.1371/journal.pone.0260554 doi: 10.1371/journal.pone.0260554
[37]	G. Zhou, J. Wu, C. Gu, B. Wang, E. D. Abel, A. K. Cheung, et al., Prorenin independently causes hypertension and renal and cardiac fibrosis in cyp1a1-prorenin transgenic rats, Clin. Sci., 132 (2018), 1345–1363. https://doi.org/10.1042/CS20171659 doi: 10.1042/CS20171659
[38]	R. Liao, B. Xie, J. Cui, Z. Qi, S. Xue, Y. Wang, E2F transcription factor 1 (E2F1) promotes the transforming growth factor TGF-β1 induced human cardiac fibroblasts differentiation through promoting the transcription of CCNE2 gene, Bioengineered, 12 (2021), 6869–6877. https://doi.org/10.1080/21655979.2021.1972194 doi: 10.1080/21655979.2021.1972194
[39]	R. Liao, Z. Qi, R. Tang, R. Wang, Y. Wang, Methyl ferulic acid attenuates human cardiac fibroblasts differentiation and myocardial fibrosis by suppressing pRB-E2F1/CCNE2 and RhoA/ROCK2 pathway, Front. Pharmacol. 12 (2021), 714390. https://doi.org/10.3389/fphar.2021.714390 doi: 10.3389/fphar.2021.714390
[40]	N. Wu, J. Xu, W. W. Du, X. Li, F. M. Awan, F. Li, et al., YAP circular RNA, circYap, attenuates cardiac fibrosis via binding with tropomyosin-4 and gamma-actin decreasing actin polymerization, Mol. Ther., 29 (2021), 1138–1150. https://doi.org/10.1016/j.ymthe.2020.12.004 doi: 10.1016/j.ymthe.2020.12.004
[41]	S. D. Prabhu, N. G. Frangogiannis, The biological basis for cardiac repair after myocardial infarction: from inflammation to fibrosis, Circ. Res., 119 (2016), 303577. https://doi.org/10.1161/CIRCRESAHA.116.303577 doi: 10.1161/CIRCRESAHA.116.303577
[42]	S. Park, N. B. Nguyen, A. Pezhouman, R. Ardehali, Cardiac fibrosis: potential therapeutic targets, Transl. Res., 209 (2019), 121–137. https://doi.org/10.1016/j.trsl.2019.03.001 doi: 10.1016/j.trsl.2019.03.001
[43]	W. Xia, H. Chen, D. Chen, Y. Ye, C. Xie, M. Hou, PD-1 inhibitor inducing exosomal miR-34a-5p expression mediates the cross talk between cardiomyocyte and macrophage in immune checkpoint inhibitor-related cardiac dysfunction, J. Immunother. Cancer, 8 (2020). https://doi.org/10.1136/jitc-2020-001293 doi: 10.1136/jitc-2020-001293
[44]	S. N. Chen, R. Lombardi, J. Karmouch, J. Y. Tsai, G. Czernuszewicz, M. R. G. Taylor, et al., DNA damage response/TP53 pathway is activated and contributes to the pathogenesis of dilated cardiomyopathy associated with LMNA (Lamin A/C) Mutations, Circ. Res., 124 (2019), 856–873. https://doi.org/10.1161/CIRCRESAHA.118.314238 doi: 10.1161/CIRCRESAHA.118.314238
[45]	A. K. Z. Johansen, J. D. Molkentin, Hippo signaling does it again: arbitrating cardiac fibroblast identity and activation, Genes Dev., 33 (2019), 1457–1459. https://doi.org/10.1101/gad.332791.119 doi: 10.1101/gad.332791.119
[46]	M. Xin, Y. Kim, L. B. Sutherland, M. Murakami, X. Qi, J. McAnally, et al., Hippo pathway effector Yap promotes cardiac regeneration, Proc. Natl. Acad. Sci. U. S. A., 110 (2013), 13839–13844. https://doi.org/10.1073/pnas.1313192110 doi: 10.1073/pnas.1313192110
[47]	N. M. Landry, S. G. Rattan, K. L. Filomeno, T. W. Meier, S. C. Meier, S. J. Foran, et al., SKI activates the Hippo pathway via LIMD1 to inhibit cardiac fibroblast activation, Basic Res. Cardiol., 116 (2021), 25. https://doi.org/10.1007/s00395-021-00865-9 doi: 10.1007/s00395-021-00865-9

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