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Transcriptome analysis reveals the spinal expression profiles of non-coding RNAs involved in anorectal malformations in rat fetuses

      Abstract

      Background

      Despite improvements in anorectal malformation (ARM) therapy, patients might still experience post-operative problems such as fecal incontinence, constipation, and soiling. In particular, the dysplasia of the lumbosacral spinal cord in ARM patients is a major disorder that affects fecal function post-operation. However, the pathological mechanisms involved are still unclear.

      Methods

      The non-coding RNAs (ncRNAs) in the lumbosacral spinal cord of fetal rats with ethylenethiourea-induced ARM were identified using RNA sequencing (RNA-seq) and examined to determine their potential function. The lumbosacral spinal cord was isolated on embryonic day 17 for subsequent RNA extraction and RNA-seq. The transcriptome data was analyzed using bioinformatics analysis, followed by validation using quantitative reverse transcription PCR.

      Results

      Compared to the control group, 26 differentially expressed microRNAs (DEMs; 22 upregulated, 4 downregulated) and 112 differentially expressed long non-coding RNAs (63 upregulated, 49 downregulated) were identified in the ARM group. Several DEMs related to development, namely miR-200a-3p, miR-200b-3p, miR-200c-3p, miR-200a-5p, and miR-429, were selected for further analysis. Notably, compared to the control, the relative expression of miR-200 family members was highly upregulated in ARM fetal rats. Furthermore, GO and KEGG enrichment and miRNA-transcription factor-lncRNA/mRNA network analysis was explored to show molecular mechanism underlying DEMs.

      Conclusions

      Our findings suggest the involvement of ncRNAs, especially the miR-200 family members, in the pathogenesis of lumbosacral spinal cord dysplasia in ARM fetal rats.

      Keywords

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      References

        • Kovacic K.
        • Matta S.R.
        • Kovacic K.
        • Calkins C.
        • Yan K.
        • Sood M.R.
        Healthcare Utilization and Comorbidities Associated with Anorectal Malformations in the United States.
        J Pediatr. 2018; 194: 142-146
        • Nam S.H.
        • Kim D.Y.
        • Kim S.C.
        Can we expect a favorable outcome after surgical treatment for an anorectal malformation?.
        J Pediatr Surg. 2016; 51: 421-424
        • Wigander H.
        • Nisell M.
        • Frenckner B.
        • Wester T.
        • Brodin U.
        • Öjmyr-Joelsson M.
        Quality of life and functional outcome in Swedish children with low anorectal malformations: a follow-up study.
        Pediatr Surg Int. 2019; 35: 583-590
        • Yang Z.
        • Geng Y.
        • Yao Z.
        • Jia H.
        • Bai Y.
        • Wang W.
        Spatiotemporal Expression of Bcl-2/Bax and Neural Cell Apoptosis in the Developing Lumbosacral Spinal Cord of Rat Fetuses with Anorectal Malformations.
        Neurochem Res. 2017; 42: 3160-3169
        • Yang Z.
        • Gao L.
        • Jia H.
        • Bai Y.
        • Wang W.
        The expression of Shh, Ptch1, and Gli1 in the developing caudal spinal cord of fetal rats with ARMs.
        J Surg Res. 2019; 233: 173-182
        • Pauli A.
        • Rinn J.L.
        • Schier A.F.
        Non-coding RNAs as regulators of embryogenesis.
        Nat Rev Genet. 2011; 12: 136-149
        • Qu Y.
        • Liu D.
        • Jia H.
        • Yang Z.
        Circular RNA rno_circ_0004002 regulates cell proliferation, apoptosis, and epithelial-mesenchymal transition through targeting miR-342-5p and Wnt3a in anorectal malformations.
        J Cell Biochem. 2019; 120
        • Jin S.
        • Wang J.
        • Chen H.
        • Xiang B.
        Differential miRNA expression analysis during late stage terminal hindgut development in fetal rats.
        J Pediatr Surg. 2017; 52: 1516-1519
        • Xiao H.
        • Huang R.
        • Chen L.
        • Diao M.
        • Li L.
        Integrating lncRNAs and mRNAs expression profiles in terminal hindgut of fetal rats with anorectal malformations.
        Pediatr Surg Int. 2018; 34: 971-982
        • Hu Z.
        • Li Z.
        miRNAs in synapse development and synaptic plasticity.
        Curr Opin Neurobiol. 2017; 45: 24-31
        • Schmitz S.U.
        • Grote P.
        • Herrmann B.G.
        Mechanisms of long noncoding RNA function in development and disease.
        Cell Mol Life Sci. 2016; 73: 2491-2509
        • Li H.
        seqtk Toolkit for processing sequences in FASTA/Q formats.
        GitHub. 2012; 767: 69
        • Kim D.
        • Paggi J.M.
        • Park C.
        • Bennett C.
        • Salzberg S.L.
        Graph-based genome alignment and genotyping with HISAT2 and HISAT-genotype.
        Nat Biotechnol. 2019; 37: 907-915
        • Robinson M.D.
        • McCarthy D.J.
        • Smyth G.K.
        edgeR: a Bioconductor package for differential expression analysis of digital gene expression data.
        Bioinformatics. 2010; 26: 139-140
        • Pertea G.
        • Pertea M.
        GFF Utilities: GffRead and GffCompare. 2020; 9 (F1000Res)
        • Tafer H.
        • Hofacker I.L.
        RNAplex: a fast tool for RNA-RNA interaction search.
        Bioinformatics. 2008; 24: 2657-2663
        • Krek A.
        • Grün D.
        • Poy M.N.
        • Wolf R.
        • Rosenberg L.
        • Epstein E.J.
        • et al.
        Combinatorial microRNA target predictions.
        Nat Genet. 2005; 37: 495-500
        • Lewis B.P.
        • Burge C.B.
        • Bartel D.P.
        Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets.
        Cell. 2005; 120: 15-20
        • Shannon P.
        • Markiel A.
        • Ozier O.
        • Baliga N.S.
        • Wang J.T.
        • Ramage D.
        • et al.
        Cytoscape: a software environment for integrated models of biomolecular interaction networks.
        Genome Res. 2003; 13: 2498-2504
        • Kanehisa M.
        • Goto S.
        KEGG: kyoto encyclopedia of genes and genomes.
        Nucleic Acids Res. 2000; 28: 27-30
        • Xie C.
        • Mao X.
        • Huang J.
        • Ding Y.
        • Wu J.
        • Dong S.
        • et al.
        KOBAS 2.0: a web server for annotation and identification of enriched pathways and diseases.
        Nucleic Acids Res. 2011; 39 (Web Server issue): W316-W322
        • Mi J.
        • Chen D.
        • Ren X.
        • Jia H.
        • Gao H.
        • Wang W.
        Spatiotemporal expression of Wnt5a during the development of the striated muscle complex in rats with anorectal malformations.
        Int J Clin Exp Pathol. 2014; 7: 1997-2005
        • Shimojo M.
        • Madara J.
        • Pankow S.
        • Liu X.
        • Yates J.
        • Südhof T.C.
        • et al.
        Synaptotagmin-11 mediates a vesicle trafficking pathway that is essential for development and synaptic plasticity.
        Genes Dev. 2019; 33 (3rd): 365-376
        • Kirmse K.
        • Hübner C.A.
        • Isbrandt D.
        • Witte O.W.
        • Holthoff K.
        GABAergic Transmission during Brain Development: multiple Effects at Multiple Stages.
        Neuroscientist. 2018; 24: 36-53
        • Long C.Y.
        • Tang X.B.
        • Wang W.L.
        • Yuan Z.W.
        • Bai Y.Z.
        Microarray analysis of miRNAs during hindgut development in rat embryos with ethylenethiourea‑induced anorectal malformations.
        Int J Mol Med. 2018; 42: 2363-2372
        • Xiao H.
        • Huang R.
        • Diao M.
        • Li L.
        • Cui X.
        Integrative analysis of microRNA and mRNA expression profiles in fetal rat model with anorectal malformation.
        PeerJ. 2018; 6: e5774
        • Yuan Z.W.
        • Lui V.C.
        • Tam P.K.
        Deficient motor innervation of the sphincter mechanism in fetal rats with anorectal malformation: a quantitative study by fluorogold retrograde tracing.
        J Pediatr Surg. 2003; 38: 1383-1388
        • Jia H.
        • Zhang K.
        • Zhang S.
        • Yuan Z.
        • Bai Y.
        • Wang W.
        Quantitative analysis of sacral parasympathetic nucleus innervating the rectum in rats with anorectal malformation.
        J Pediatr Surg. 2007; 42: 1544-1548
        • Trümbach D.
        • Prakash N.
        The conserved miR-8/miR-200 microRNA family and their role in invertebrate and vertebrate neurogenesis.
        Cell Tissue Res. 2015; 359: 161-177
        • Pandey A.
        • Singh P.
        • Jauhari A.
        • Singh T.
        • Khan F.
        • Pant A.B.
        • et al.
        Critical role of the miR-200 family in regulating differentiation and proliferation of neurons.
        J Neurochem. 2015; 133: 640-652
        • Buller B.
        • Chopp M.
        • Ueno Y.
        • Zhang L.
        • Zhang R.L.
        • Morris D.
        • et al.
        Regulation of serum response factor by miRNA-200 and miRNA-9 modulates oligodendrocyte progenitor cell differentiation.
        Glia. 2012; 60: 1906-1914
        • Jiang Q.
        • Wang Y.
        • Shi X.
        Propofol Inhibits Neurogenesis of Rat Neural Stem Cells by Upregulating MicroRNA-141-3p.
        Stem Cells Dev. 2017; 26: 189-196
        • Fu J.
        • Shrivastava A.
        • Shrivastava S.K.
        • Srivastava R.K.
        • Shankar S.
        Triacetyl resveratrol upregulates miRNA‑200 and suppresses the Shh pathway in pancreatic cancer: a potential therapeutic agent.
        Int J Oncol. 2019; 54: 1306-1316
        • Guan T.
        • Dominguez C.X.
        • Amezquita R.A.
        • Laidlaw B.J.
        • Cheng J.
        • Henao-Mejia J.
        • et al.
        ZEB1, ZEB2, and the miR-200 family form a counterregulatory network to regulate CD8(+) T cell fates.
        J Exp Med. 2018; 215: 1153-1168
        • Title A.C.
        • Hong S.J.
        • Pires N.D.
        • Hasenöhrl L.
        • Godbersen S.
        • Stokar-Regenscheit N.
        • et al.
        Genetic dissection of the miR-200-Zeb1 axis reveals its importance in tumor differentiation and invasion.
        Nat Commun. 2018; 9: 4671
        • Chen L.
        • Gibbons D.L.
        • Goswami S.
        • Cortez M.A.
        • Ahn Y.H.
        • Byers L.A.
        • et al.
        Metastasis is regulated via microRNA-200/ZEB1 axis control of tumour cell PD-L1 expression and intratumoral immunosuppression.
        Nat Commun. 2014; 5: 5241
        • Yan X.T.
        • Zhao Y.
        • Cheng X.L.
        • He X.H.
        • Wang Y.
        • Zheng W.Z.
        • et al.
        Inhibition of miR-200b/miR-429 contributes to neuropathic pain development through targeting zinc finger E box binding protein-1.
        J Cell Physiol. 2018; 233: 4815-4824
        • Antonello Z.A.
        • Reiff T.
        • Ballesta-Illan E.
        • Dominguez M.
        Robust intestinal homeostasis relies on cellular plasticity in enteroblasts mediated by miR-8-Escargot switch.
        Embo j. 2015; 34: 2025-2041
        • Qiu J.
        • Ma X.
        • Zeng F.
        • Yan J.
        RNA editing regulates lncRNA splicing in human early embryo development.
        PLoS Comput Biol. 2021; 17e1009630
        • Bartel D.P.
        MicroRNAs: target recognition and regulatory functions.
        Cell. 2009; 136: 215-233
        • Liu J.
        • Ye X.
        • Wu F.X.
        Characterizing dynamic regulatory programs in mouse lung development and their potential association with tumourigenesis via miRNA-TF-mRNA circuits.
        BMC Syst Biol. 2013; (7 Suppl 2Suppl 2): S11
        • Lee C.J.
        • Ahn H.
        • Lee S.B.
        • Shin J.Y.
        • Park W.Y.
        • Kim J.I.
        • et al.
        Integrated analysis of omics data using microRNA-target mRNA network and PPI network reveals regulation of Gnai1 function in the spinal cord of Ews/Ewsr1 KO mice.
        BMC Med Genomics. 2016; (9 Suppl 1Suppl 1)): 33
        • Jia S.
        • Zhang Q.
        • Wang Y.
        • Wang Y.
        • Liu D.
        • He Y.
        • et al.
        PIWI-interacting RNA sequencing profiles in maternal plasma-derived exosomes reveal novel non-invasive prenatal biomarkers for the early diagnosis of nonsyndromic cleft lip and palate.
        EBioMedicine. 2021; 65103253
        • Gu H.
        • Chen L.
        • Xue J.
        • Huang T.
        • Wei X.
        • Liu D.
        • et al.
        Expression profile of maternal circulating microRNAs as non-invasive biomarkers for prenatal diagnosis of congenital heart defects.
        Biomed Pharmacother. 2019; 109: 823-830
        • Gu H.
        • Li H.
        • Zhang L.
        • Luan H.
        • Huang T.
        • Wang L.
        • et al.
        Diagnostic role of microRNA expression profile in the serum of pregnant women with fetuses with neural tube defects.
        J Neurochem. 2012; 122: 641-649
        • Sun P.
        • Liu D.Z.
        • Jickling G.C.
        • Sharp F.R.
        • Yin K.J.
        MicroRNA-based therapeutics in central nervous system injuries.
        J Cereb Blood Flow Metab. 2018; 38: 1125-1148
        • Ma W.
        • Wei X.
        • Gu H.
        • Liu D.
        • Luo W.
        • An D.
        • et al.
        Therapeutic potential of adenovirus-encoding brain-derived neurotrophic factor for spina bifida aperta by intra-amniotic delivery in a rat model.
        Gene Ther. 2020; 27: 567-578
        • Wei X.
        • Cao S.
        • Ma W.
        • Zhang C.
        • Gu H.
        • Liu D.
        • et al.
        Intra-Amniotic Delivery of CRMP4 siRNA Improves Mesenchymal Stem Cell Therapy in a Rat Spina Bifida Model.
        Mol Ther Nucleic Acids. 2020; 20: 502-517