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Microarray Expression Profile of Mrnas and Long Noncoding Rnas

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Microarray Expression Profile of Mrnas and Long Noncoding Rnas Yang et al. Cell Div (2021) 16:1 https://doi.org/10.1186/s13008-020-00069-y Cell Division RESEARCH Open Access Microarray expression profle of mRNAs and long noncoding RNAs and the potential role of PFK-1 in infantile hemangioma Kaiying Yang1, Xuepeng Zhang2, Linwen Chen3, Siyuan Chen2* and Yi Ji1* Abstract Background: Infantile hemangioma (IH) is the most common benign tumor in children. Long noncoding RNAs (lncRNAs) play a critical role in tumorigenesis. However, the expression levels and biological functions of lncRNAs in IH have not been well-studied. This study aimed to analyze the expression profle of lncRNAs and mRNAs in proliferating and involuting IHs. Methods: The expression profles of lncRNAs and mRNAs in proliferating and involuting IHs were identifed by micro- array analysis. Subsequently, detailed bioinformatics analyses were performed. Finally, quantitative real-time polymer- ase chain reaction (qRT-PCR) and immunohistochemistry (IHC) analyses were conducted to validate the microarray results. Results: In total, 146 diferentially expressed (DE) lncRNAs and 374 DE mRNAs were identifed. The DE mRNAs were enriched mostly in angiogenesis-related biological processes (BPs) and pathways by bioinformatics analysis. In addi- tion, metabolism-related BPs (e.g., “glycogen biosynthetic process” and “metabolic process”) and pathways (e.g., “oxida- tive phosphorylation”) were identifed. A lncRNA-mRNA co-expression network was constructed from 42 DE lncRNAs and 217 DE mRNAs. Twelve lncRNAs were predicted to have cis-regulated target genes. The microarray analysis results were validated by qRT-PCR using 5 randomly selected lncRNAs and 13 mRNAs. The IHC results revealed that both LOXL2 and FPK-1 exhibited higher protein expression levels in proliferating IH than in involuting IH. Moreover, inhibi- tion of PFK-1 could suppress hemangioma-derived endothelial cell proliferation and migration, induce cell arrest, and reduce glucose uptake and lactate and ATP production. Conclusions: The fndings suggest that the identifed DE lncRNAs and mRNAs may be associated with the patho- genesis of IH. The data presented herein can improve our understanding of IH development and provide direction for further studies investigating the mechanism underlying IH. Keywords: Infantile hemangioma, Long noncoding RNA, mRNA, PFK-1, Microarray, Bioinformatics analysis Introduction Infantile hemangioma (IH) is the most common benign tumor in children, with a prevalence of 4–5% [1]. IH is predominantly found in female children; the female:male *Correspondence: [email protected]; [email protected] 1 Division of Oncology, Department of Pediatric Surgery, West China ratio ranges from 1.4:1 to 3:1 [2]. IH is usually absent Hospital of Sichuan University, #37 Guo-Xue-Xiang, Chengdu 610041, at birth and exhibits a characteristic growth pattern China with a proliferating phase lasting for 1 year after birth, 2 Pediatric Intensive Care Unit, Department of Critical Care Medicine, West China Hospital of Sichuan University, Chengdu 610041, China with the most rapid growth at 5–8 weeks, followed by Full list of author information is available at the end of the article spontaneous involution lasting up to 5 years [3]. In the © The Author(s) 2021. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/publi cdoma in/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Yang et al. Cell Div (2021) 16:1 Page 2 of 17 proliferating phase, the IH will enlarge, become more ele- of all patients. All IH tissues were obtained surgically at vated and develop a rubbery consistency; in contrast, in the West China Hospital of Sichuan University. In total, the involuting phase, the IH will fatten and shrink from tissues from six proliferating IHs and four involuting IHs the center outward [4]. IHs are most frequently found in were collected, and all specimens were stored at − 80 °C the head and neck, followed by the trunk and extremi- after excision. Detailed information on the ten patients is ties. Indications for treatment include disfgurement, presented in Additional fle 1: Table S1. impaired function and/or a threat to life [5]. Both vasculogenesis and angiogenesis are widely Cell culture accepted to contribute to the mechanism of IH develop- Hemangioma-derived endothelial cell (HemEC) isola- ment [6]. Several signaling pathways that regulate vascu- tion was performed as described previously [16–18]. Te logenesis and angiogenesis have been demonstrated to HemECs were cultured with Endothelial basal medium be associated with the pathogenesis of IH [7, 8] primarily (EBM-2, Lonza, Walkersville, MD, USA) containing 10% including the vascular endothelial growth factor (VEGF) fetal bovine serum (Gibco), SingleQuot (Lonza), peni- and VEGF receptor pathways, notch pathway, mamma- cillin (Gibco) and streptomycin (Gibco). Te cells were lian target of rapamycin pathway, β-adrenergic signaling grown in a humidifed atmosphere containing 5% CO2 in pathway and angiopoietin and Tie2 signaling pathway [7] the air at 37 °C. [8],. However, the exact pathogenesis of IH has not been fully elucidated. RNA extraction Long noncoding RNAs (lncRNAs) are most commonly Total RNA was extracted from frozen tissues using the defned as transcripts longer than 200 nucleotides with- TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and was out protein-coding function [9]. LncRNAs were pre- further purifed with a miRNeasy Mini Kit (QIAGEN, viously considered transcriptional “noise”. However, Valencia, CA, USA) according to the manufacturer’s accumulated studies indicate that lncRNAs play a critical instructions. RNA quantity and quality were measured role in multiple biological processes (BPs), including the with a NanoDrop 2000 spectrophotometer (Termo regulation of gene expression at the chromatin modifca- Scientifc, Wilmington, DE, USA), and RNA purity and tion level, transcriptional and posttranscriptional pro- integrity were assessed by agarose gel electrophoresis. cessing, and cell diferentiation and development [9, 10]. Ten, RNA samples were stored at -80 °C until further Recently, lncRNAs have attracted increasing attention use. in tumorigenesis for their important roles in stemness acquisition and maintenance, growth and metastasis of Microarray analysis cancer cells, as well as in vascular diseases [11]. Moreo- LncRNA and mRNA expression profling were per- ver, lncRNAs have been demonstrated to play a critical formed using the Afymetrix GeneChip Human Tran- role in endothelial cell (EC) diferentiation and angiogen- scriptome Array 2.0 platform (Afymetrix, Inc., Santa esis [12]. Clara, CA, USA). Tis high-resolution array contains Several studies have reported that lncRNAs are impli- more than 6 million distinct probes, including over cated in IH [13–15]. However, the association between 245,000 mRNA and 40,000 lncRNA probes. Raw micro- IH and the expression levels and biological functions array data were extracted by using the Agilent Feature of lncRNAs remains unclear. Tis study aimed to ana- Extraction (v. 10.7), summarized, normalized and qual- lyze the expression profle of lncRNAs and mRNAs in ity-controlled using the GeneSpring GX program soft- proliferating and involuting IH by microarray analy- ware (v. 12.6.1) package (Agilent Technologies) and R sis. Additionally, using Gene Ontology (GO) and Kyoto program. Ten microarray analysis was performed via Encyclopedia of Genes and Genomes (KEGG) pathway the Gene-Cloud of Biotechnology Information (GCBI, analysis, we identifed the clinical signifcance of diferen- Shanghai, China; https ://www.gcbi.com.cn) platform. tially expressed (DE) lncRNAs and mRNAs and validated Briefy, the DE lncRNAs and mRNAs between the pro- their expression using quantitative real-time polymerase liferating group and the involuting group were frst chain reaction (qRT-PCR) and immunohistochemistry identifed. Second, GO enrichment and KEGG pathway (IHC) staining. analyses were performed with the DE genes. Finally, the DE lncRNAs and mRNAs were used to construct a Materials and methods lncRNA-mRNA co-expression network. LncRNAs and Samples mRNAs signifcantly DE between the two groups were Tis study was approved by the Institutional Review identifed by fltering with an adjusted P value < 0.05 and Board of the West China Hospital of Sichuan University. fold change > 1.5 using SAM analysis. A volcano plot was Written informed consent was obtained from the parents generated to distinguish the signifcantly DE lncRNAs Yang et al. Cell Div (2021) 16:1 Page 3 of 17 and mRNAs. Hierarchical clustering was applied to dis- qRT‑PCR validation play the expression profles of signifcantly DE lncRNAs qRT-PCR analysis was performed using SYBR qPCR and mRNAs between the two groups. Master Mix (Vazyme, Nanjing, China) according to the manufacturer’s protocol. Te qRT-PCR conditions were GO and KEGG pathway analyses as follows: 95 °C for 30 s, followed by 40 cycles at 95 °C for 15 s and 60 °C for 30 s. All experiments were per- GO analysis was used to categorize and describe the bio- formed in triplicate. Te expression levels of lncRNAs logical functions of the signifcantly DE genes using the ΔΔCt and mRNAs were quantifed using the 2− method cellular component (CC), molecular function (MF) and and normalized to GAPDH expression. All the prim- BP categories.
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