Login Register Cart 0
General Research Article

High-Throughput Sequencing Reveals N6-Methyladenosine-modified LncRNAs as Potential Biomarkers in Mice with Liver Fibrosis

Author(s): Furong Wu, Shengyu Zhang, Chang Fan, Shaopeng Huang, Hui Jiang* and Jiafu Zhang*

Volume 23, Issue 5, 2023

Published on: 03 July, 2023

Page: [371 - 390] Pages: 20

DOI: 10.2174/1566523223666230606151013

Price: $65

conference banner
Abstract

Background: N6-methyladenosine (m6A) is the most frequent internal modification in eukaryotic RNA. Long noncoding RNAs (lncRNAs) are a new type of noncoding regulatory molecule with multiple cellular functions. Both are closely related to the occurrence and development of liver fibrosis (LF). However, the role of m6A-methylated lncRNAs in the progression of LF remains largely unknown.

Methods: In this study, HE and Masson staining were used to observe pathological changes in the liver, m6A-modified RNA immunoprecipitation sequencing (m6A-seq) was performed to systematically evaluate the m6A modification level of lncRNAs in LF mice, meRIP-qPCR and RT-qPCR were used to detect the m6A methylation level and RNA expression level of the target lncRNAs.

Results: A total of 415 m6A peaks were detected in 313 lncRNAs in liver fibrosis tissues. There were 98 significantly different m6A peaks in LF, which were located on 84 lncRNAs, of which 45.2% of the lncRNA length was between 200-400 bp. At the same time, the first three chromosomes of these methylated lncRNAs were chromosomes 7, 5 and 1. RNA sequencing identified 154 differentially expressed lncRNAs in LF. The joint analysis of m6A-seq and RNA-seq found that there were three lncRNAs with significant changes in m6A methylation and RNA expression levels: lncRNA H19, lncRNA Gm16023 and lncRNA Gm17586. Subsequently, the verification results showed that the m6A methylation levels of lncRNA H19 and lncRNA Gm17586 were significantly increased, while that of lncRNA Gm16023 was significantly decreased, and the RNA expression of three lncRNAs was significantly decreased. Through the establishment of a lncRNA-miRNA-mRNA regulatory network, the possible regulatory relationships of lncRNA H19, lncRNA Gm16023 and lncRNA Gm17586 in LF were revealed.

Conclusion: This study revealed the unique m6A methylation pattern of lncRNAs in LF mice, suggesting that the m6A methylation modification of lncRNAs is related to the occurrence and development of LF.

Keywords: N6-methyladenosine, lncRNA, regulatory network, liver fibrosis, high-throughput sequencing, meRIP-qPCR.

« Previous Next »
Graphical Abstract

[1]
Song M, Pebworth MP, Yang X, et al. Cell-type-specific 3D epigenomes in the developing human cortex. Nature 2020; 587(7835): 644-9.
[http://dx.doi.org/10.1038/s41586-020-2825-4] [PMID: 33057195]
[2]
Tanakas S, Aubin JE, van Wijnen AJ. Epigenetic control of skeletal homeostasis and diseases. Bone 2021; 144: 115797.
[http://dx.doi.org/10.1016/j.bone.2020.115797] [PMID: 33333242]
[3]
Ohkura N, Yasumizu Y, Kitagawa Y, et al. Regulatory T cell-specific epigenomic region variants are a key determinant of susceptibility to common autoimmune diseases. Immunity 2020; 52(6): 1119-1132.e4.
[http://dx.doi.org/10.1016/j.immuni.2020.04.006] [PMID: 32362325]
[4]
Zhang H, Shi X, Huang T, et al. Dynamic landscape and evolution of m6A methylation in human. Nucleic Acids Res 2020; 48(11): 6251-64.
[http://dx.doi.org/10.1093/nar/gkaa347] [PMID: 32406913]
[5]
Ma Z, Gao X, Shuai Y, Xing X, Ji J. The m6A epitranscriptome opens a new charter in immune system logic. Epigenetics 2021; 16(8): 819-37.
[http://dx.doi.org/10.1080/15592294.2020.1827722] [PMID: 33070685]
[6]
Li E, Wei B, Wang X, Kang R. METTL3 enhances cell adhesion through stabilizing integrin β1 mRNA via an m6A-HuR-dependent mechanism in prostatic carcinoma. Am J Cancer Res 2020; 10(3): 1012-25.
[PMID: 32266107]
[7]
Du YD, Guo WY, Han CH, et al. N6-methyladenosine demethylase FTO impairs hepatic ischemia–reperfusion injury via inhibiting Drp1-mediated mitochondrial fragmentation. Cell Death Dis 2021; 12(5): 442.
[http://dx.doi.org/10.1038/s41419-021-03622-x] [PMID: 33947842]
[8]
Liu J, Harada BT, He C. Regulation of gene expression by N-methyladenosine in Cancer. Trends Cell Biol 2019; 29(6): 487-99.
[http://dx.doi.org/10.1016/j.tcb.2019.02.008] [PMID: 30940398]
[9]
Yue C, Chen J, Li Z, Li L, Chen J, Guo Y. microRNA-96 promotes occurrence and progression of colorectal cancer via regulation of the AMPKα2-FTO-m6A/MYC axis. J Exp Clin Cancer Res 2020; 39(1): 240.
[http://dx.doi.org/10.1186/s13046-020-01731-7] [PMID: 33183350]
[10]
Guo X, Li K, Jiang W, et al. RNA demethylase ALKBH5 prevents pancreatic cancer progression by posttranscriptional activation of PER1 in an m6A-YTHDF2-dependent manner. Mol Cancer 2020; 19(1): 91.
[http://dx.doi.org/10.1186/s12943-020-01158-w] [PMID: 32429928]
[11]
Shi R, Ying S, Li Y, Zhu L, Wang X, Jin H. Linking the YTH domain to cancer: The importance of YTH family proteins in epigenetics. Cell Death Dis 2021; 12(4): 346.
[http://dx.doi.org/10.1038/s41419-021-03625-8] [PMID: 33795663]
[12]
Zhu L, Li S, He S, et al. The critical role of m6A methylation in the pathogenesis of Graves’ ophthalmopathy. Eye Vis 2020; 7(1): 55.
[http://dx.doi.org/10.1186/s40662-020-00221-3] [PMID: 33292635]
[13]
Xue M, Zhao BS, Zhang Z, et al. Viral N6-methyladenosine upregulates replication and pathogenesis of human respiratory syncytial virus. Nat Commun 2019; 10(1): 4595.
[http://dx.doi.org/10.1038/s41467-019-12504-y] [PMID: 31597913]
[14]
Xiang S, Liang X, Yin S, Liu J, Xiang Z. N6-methyladenosine methyltransferase METTL3 promotes colorectal cancer cell proliferation through enhancing MYC expression. Am J Transl Res 2020; 12(5): 1789-806.
[PMID: 32509177]
[15]
Wang Y, Zheng Y, Guo D, et al. m6A methylation analysis of differentially expressed genes in skin tissues of coarse and fine type liaoning cashmere goats. Front Genet 2020; 10: 1318.
[http://dx.doi.org/10.3389/fgene.2019.01318] [PMID: 32038703]
[16]
Han Z, Wang X, Xu Z, et al. ALKBH5 regulates cardiomyocyte proliferation and heart regeneration by demethylating the mRNA of YTHDF1. Theranostics 2021; 11(6): 3000-16.
[http://dx.doi.org/10.7150/thno.47354] [PMID: 33456585]
[17]
He S, Wang H, Liu R, et al. mRNA N6-methyladenosine methylation of postnatal liver development in pig. PLoS One 2017; 12(3): e0173421.
[http://dx.doi.org/10.1371/journal.pone.0173421] [PMID: 28267806]
[18]
Barajas JM, Lin CH, Sun HL, et al. METTL3 regulates liver homeostasis, hepatocyte ploidy, and circadian Rhythm-controlled gene expression in mice. Am J Pathol 2022; 192(1): 56-71.
[PMID: 34599880]
[19]
Zhou B, Liu C, Xu L, et al. N6-Methyladenosine reader protein YT521-B homology domain-containing 2 suppresses liver steatosis by regulation of mRNA stability of lipogenic genes. Hepatology 2021; 73(1): 91-103.
[http://dx.doi.org/10.1002/hep.31220] [PMID: 32150756]
[20]
Shi Z, Zhang K, Chen T, et al. Transcriptional factor ATF3 promotes liver fibrosis via activating hepatic stellate cells. Cell Death Dis 2020; 11(12): 1066.
[http://dx.doi.org/10.1038/s41419-020-03271-6] [PMID: 33311456]
[21]
Zong Z, Liu J, Wang N, et al. Nicotinamide mononucleotide inhibits hepatic stellate cell activation to prevent liver fibrosis via promoting PGE2 degradation. Free Radic Biol Med 2021; 162: 571-81.
[http://dx.doi.org/10.1016/j.freeradbiomed.2020.11.014] [PMID: 33220424]
[22]
Hong F, Wan L, Liu J, et al. Histone methylation regulates Hif-1 signaling cascade in activation of hepatic stellate cells. FEBS Open Bio 2018; 8(3): 406-15.
[http://dx.doi.org/10.1002/2211-5463.12379] [PMID: 29511617]
[23]
Rinaldi L, Ascione A, Messina V, et al. Influence of antiviral therapy on the liver stiffness in chronic HBV hepatitis. Infection 2018; 46(2): 231-8.
[http://dx.doi.org/10.1007/s15010-017-1113-1] [PMID: 29335905]
[24]
Cui Z, Huang N, Liu L, et al. Dynamic analysis of m6A methylation spectroscopy during progression and reversal of hepatic fibrosis. Epigenomics 2020; 12(19): 1707-23.
[http://dx.doi.org/10.2217/epi-2019-0365] [PMID: 33174480]
[25]
Zhu Y, Pan X, Du N, et al. ASIC1a regulates miR-350/SPRY2 by N6-methyladenosine to promote liver fibrosis. FASEB J 2020; 34(11): 14371-88.
[http://dx.doi.org/10.1096/fj.202001337R] [PMID: 32949431]
[26]
Lu L, Zhang Y, He Q, et al. MTA, an RNA m6A methyltransferase, enhances drought tolerance by regulating the development of trichomes and roots in poplar. Int J Mol Sci 2020; 21(7): 2462.
[http://dx.doi.org/10.3390/ijms21072462] [PMID: 32252292]
[27]
Zheng N, Su J, Hu H, Wang J, Chen X. Research progress of N6-methyladenosine in the cardiovascular system. Med Sci Monit 2020; 26: e921742.
[http://dx.doi.org/10.12659/MSM.921742] [PMID: 32350237]
[28]
He Z, Yang D, Fan X, et al. The roles and mechanisms of lncRNAs in liver fibrosis. Int J Mol Sci 2020; 21(4): 1482.
[http://dx.doi.org/10.3390/ijms21041482] [PMID: 32098245]
[29]
Barth DA, Prinz F, Teppan J, Jonas K, Klec C, Pichler M. Long-Noncoding RNA (lncRNA) in the regulation of Hypoxia-Inducible Factor (HIF) in cancer. Noncoding RNA 2020; 6(3): 27.
[http://dx.doi.org/10.3390/ncrna6030027] [PMID: 32640630]
[30]
Nolte W, Weikard R, Brunner RM, et al. Biological network approach for the identification of regulatory long non-coding RNAs associated with metabolic efficiency in cattle. Front Genet 2019; 10: 1130.
[http://dx.doi.org/10.3389/fgene.2019.01130] [PMID: 31824560]
[31]
Mao C, Wang X, Liu Y, et al. A G3BP1-interacting lncRNA promotes ferroptosis and apoptosis in cancer via nuclear sequestration of p53. Cancer Res 2018; 78(13): 3484-96.
[http://dx.doi.org/10.1158/0008-5472.CAN-17-3454] [PMID: 29588351]
[32]
Fan C, Ma Y, Chen S, et al. Comprehensive analysis of the transcriptome-wide m6A methylation modification difference in liver fibrosis mice by high-throughput m6A sequencing. Front Cell Dev Biol 2021; 9: 767051.
[http://dx.doi.org/10.3389/fcell.2021.767051] [PMID: 34869362]
[33]
Fan C, Wu FR, Zhang JF, Jiang H. A network pharmacology approach to explore the mechanisms of shugan jianpi formula in liver fibrosis. Evid Based Complement Alternat Med 2020; 2020: 4780383.
[http://dx.doi.org/10.1155/2020/4780383]
[34]
Liang Q, Li X, Guan G, et al. Long non-coding RNA, HOTAIRM1, promotes glioma malignancy by forming a ceRNA network. Aging 2019; 11(17): 6805-38.
[http://dx.doi.org/10.18632/aging.102205] [PMID: 31477638]
[35]
Chen X, Lv C, Zhu X, et al. MicroRNA-504 modulates osteosarcoma cell chemoresistance to cisplatin by targeting p53. Oncol Lett 2019; 17(2): 1664-74.
[PMID: 30675226]
[36]
Ye J, Lin Y, Yu Y, Sun D. LncRNA NEAT1/microRNA-129-5p/SOCS2 axis regulates liver fibrosis in alcoholic steatohepatitis. J Transl Med 2020; 18(1): 445.
[http://dx.doi.org/10.1186/s12967-020-02577-5] [PMID: 33228663]
[37]
Zhang K, Han X, Zhang Z, et al. The liver-enriched lnc-LFAR1 promotes liver fibrosis by activating TGFβ and Notch pathways. Nat Commun 2017; 8(1): 144.
[http://dx.doi.org/10.1038/s41467-017-00204-4] [PMID: 28747678]
[38]
Yang JJ, Yang Y, Zhang C, Li J, Yang Y. Epigenetic silencing of LncRNA ANRIL enhances liver fibrosis and HSC activation through activating AMPK pathway. J Cell Mol Med 2020; 24(4): 2677-87.
[http://dx.doi.org/10.1111/jcmm.14987] [PMID: 31961061]
[39]
He Y, Wu Y, Huang C, et al. Inhibitory effects of long noncoding RNA MEG3 on hepatic stellate cells activation and liver fibrogenesis. Biochim Biophys Acta Mol Basis Dis 2014; 1842(11): 2204-15.
[http://dx.doi.org/10.1016/j.bbadis.2014.08.015] [PMID: 25201080]
[40]
Zuo L, Su H, Zhang Q, et al. Comprehensive analysis of lncRNAs N6-methyladenosine modification in colorectal cancer. Aging 2021; 13(3): 4182-98.
[http://dx.doi.org/10.18632/aging.202383] [PMID: 33493136]
[41]
Niu X, Xu J, Liu J, Chen L, Qiao X, Zhong M. Landscape of N6-methyladenosine modification patterns in human ameloblastoma. Front Oncol 2020; 10: 556497.
[http://dx.doi.org/10.3389/fonc.2020.556497] [PMID: 33178585]
[42]
Kasowitz SD, Ma J, Anderson SJ, et al. Nuclear m6A reader YTHDC1 regulates alternative polyadenylation and splicing during mouse oocyte development. PLoS Genet 2018; 14(5): e1007412.
[http://dx.doi.org/10.1371/journal.pgen.1007412] [PMID: 29799838]
[43]
Zhang Z, Luo K, Zou Z, et al. Genetic analyses support the contribution of mRNA N6-methyladenosine (m6A) modification to human disease heritability. Nat Genet 2020; 52(9): 939-49.
[http://dx.doi.org/10.1038/s41588-020-0644-z] [PMID: 32601472]
[44]
Zheng H, Li S, Zhang X, Sui N. Functional implications of active N6-methyladenosine in plants. Front Cell Dev Biol 2020; 8: 291.
[http://dx.doi.org/10.3389/fcell.2020.00291] [PMID: 32411708]
[45]
Dominissini D, Moshitch-Moshkovitz S, Schwartz S, et al. Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq. Nature 2012; 485(7397): 201-6.
[http://dx.doi.org/10.1038/nature11112] [PMID: 22575960]
[46]
Wu Y, Yang X, Chen Z, et al. m6A-induced lncRNA RP11 triggers the dissemination of colorectal cancer cells via upregulation of Zeb1. Mol Cancer 2019; 18(1): 87.
[http://dx.doi.org/10.1186/s12943-019-1014-2] [PMID: 30979372]
[47]
Wang J, Ding W, Xu Y, et al. Long non-coding RNA RHPN1-AS1 promotes tumorigenesis and metastasis of ovarian cancer by acting as a ceRNA against miR-596 and upregulating LETM1. Aging 2020; 12(5): 4558-72.
[http://dx.doi.org/10.18632/aging.102911] [PMID: 32163372]
[48]
Yang X, Zhang S, He C, et al. METTL14 suppresses proliferation and metastasis of colorectal cancer by down-regulating oncogenic long non-coding RNA XIST. Mol Cancer 2020; 19(1): 46.
[http://dx.doi.org/10.1186/s12943-020-1146-4] [PMID: 32111213]
[49]
Ghafouri-Fard S, Esmaeili M, Taheri M. H19 lncRNA: Roles in tumorigenesis. Biomed Pharmacother 2020; 123: 109774.
[50]
Yang JJ, Liu LP, Tao H, et al. MeCP2 silencing of LncRNA H19 controls hepatic stellate cell proliferation by targeting IGF1R. Toxicology 2016; 359-360: 39-46.
[http://dx.doi.org/10.1016/j.tox.2016.06.016] [PMID: 27350269]
[51]
Zhang Y, Liu C, Barbier O, et al. Bcl2 is a critical regulator of bile acid homeostasis by dictating Shp and lncRNA H19 function. Sci Rep 2016; 6(1): 20559.
[http://dx.doi.org/10.1038/srep20559] [PMID: 26838806]
[52]
Liu Y, Liu N, Liu Q. Constructing a ceRNA-immunoregulatory network associated with the development and prognosis of human atherosclerosis through weighted gene co-expression network analysis. Aging 2021; 13(2): 3080-100.
[http://dx.doi.org/10.18632/aging.202486] [PMID: 33460396]
[53]
Xia W, Chen H, Xie C, Hou M. Long-noncoding RNA MALAT1 sponges microRNA-92a-3p to inhibit doxorubicin-induced cardiac senescence by targeting ATG4a. Aging 2020; 12(9): 8241-60.
[http://dx.doi.org/10.18632/aging.103136] [PMID: 32384281]

Rights & Permissions Print Cite
Article Metrics
26
3
Wayfinder Image
© 2024 Bentham Science Publishers | Privacy Policy