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Downloaded Workfow-Type Data and Clinical Data from the Cancer Genome Atlas Project Yu and Zhu Biol Proced Online (2021) 23:15 https://doi.org/10.1186/s12575-021-00152-w RESEARCH Open Access N6-Methyladenosine Related Long Non-Coding RNAs and Immune Cell Infltration in the Tumor Microenvironment of Gastric Cancer Zhong lin Yu1,2 and Zheng ming Zhu1,2* Abstract Aim: To illustrate the infuence of N6-methyladenosine long non-coding RNAs and immune cell infltration in gastric cancer. Methods: We downloaded workfow-type data and clinical data from The Cancer Genome Atlas project. The relation- ship of lncRNA and m6A was identifed. Kyoto Encyclopedia of Genes and Genomes gene expression enrichment analysis was performed. Lasso regression was utilized to construct a prognostic model. Survival analysis to explore the relationship between m6A lncRNA and clinical survival data. Diferential analysis of the tumor microenvironment and immune correlation analysis to determine immune cell infltration levels and their correlation with clinical prognosis. Results: Co-expression analysis indicated that lncRNA expression was associated closely with m6A. m6A-lncRNAs were partially highly expressed in tumor tissue and could be used in a prognostic model to predict GC prognosis, independent of other clinical characteristics. “ADIPPOCYTOKINE SIGNALING PATHWAY” was most signifcantly enriched according to GSEA. ACBD3-AS1 was overexpressed in tumor tissue. Naïve B cell, Plasma cells, resting CD4 memory T cell were highly infltrated tissues in cluster 2, while Macrophages M2, resting Mast cells, Monocytes, regulates T cells were lowly in cluster 1. All related scores were higher in cluster 2, indicating a lower purity of tumor cells and higher density of immune-related cells in the tumor microenvironment. Conclusion: m6A lncRNA is closely related to the occurrence and progression of GC. The corresponding prognostic model can be utilized to evaluate the prognosis of GC. m6A lncRNA and related immune cell infltration in the tumor microenvironment can provide novel therapeutic targets for further research. Keywords: Bioinformatics analysis, N6-methyladenosine, lncRNAs, Immune cell infltration, Gastric cancer Introduction [1]. With a multidisciplinary team approach and the According to global cancer statistics, there were application of postoperative hyperthermic intraperito- 1,089,103(5.6%) new cases and 768,793(7.7%) deaths neal chemotherapy, the fve-year survival rate of gastric from gastric cancer in 2020. Te incidence and mortality cancer can be optimized to a certain extent. However, rate of gastric cancer ranked ffth and forth, respectively efective treatments for advanced metastatic GC need to be explored. One of the fundamental factors that acceler- *Correspondence: [email protected] ates the occurrence and progression of GC is the accu- 1 Department of Gastrointestinal Surgery, The Second Afliated Hospital mulation of epigenetic transformations [2]. of Nanchang University, 1 Minde Road, Nanchang 330006, Jiangxi, China Full list of author information is available at the end of the article © The Author(s) 2021. Open Access 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. Yu and Zhu Biol Proced Online (2021) 23:15 Page 2 of 18 m6A is related to several mRNA metabolism pathways cancer. gov/) up to March 2021, the public database involved in mRNA translation and decay; together with included the expression profles of 375 gastric cancer 5-methylcytosine and pseudouridine, m6A constitutes tissues and 32 normal tissues derived by HIseq-FPKM. the epitranscriptome and afects protein synthesis [3]. Ten, we built the mRNA matrix via PERL software Aberrant m6A modifcation stimulates tumor stem cell (https:// www. perl. org/) and used the corresponding self-renewal, which intricately contributes to the progres- script to organize transcriptomic data and transform gene sion of tumorigenesis [4]. Tere is increasing evidence IDs. Finally, the same software and specifc script were that long non-coding RNAs (lncRNAs) are involved in utilized to process the clinical data. When we installed a multitude of cancers based on their large number and the software, the language of the installation path was expression specifcity, and facilitating the elucidation of set as English, and there were no spaces included. Based functional cancer-associated transcripts would be ben- on the guidelines released by the National Cancer Insti- efcial for the development of therapeutic interventions tute in December 2015 (https:// cance rgeno me. nih. gov/ against cancer [5, 6]. publi catio ns/ publi cation guidelines), our research did not m6A-methylated lncRNA has important regulatory require the approval of an ethics committee. efects on several biological and pathological processes. m6A mediates immunotherapy and prolongs survival by modulating gene expression and splicing, which causes Identifcation of m6A Related lncRNAs changes in lactate levels in the tumor microenviron- To distinguish mRNAs and lncRNAs by utilizing the col- ment [7]. Furthermore, knockdown of METTL14 height- lated transcriptomic data, we constructed a gene expres- ened proliferative and invasive potential of GC cells and sion matrix and human confguration fle containing the promoted tumorigenicity and metastasis via the PI3K/ expression levels of m6A-related lncRNA gene profles AKT/mTOR signaling pathway [8]. However, the aber- using PERL software. By running the corresponding rant expression and m6A methylation of lncRNA in GC script fle, we obtained the attributes of related genes remain unknown. Terefore, the formation of a tran- and obtained the respective mRNA and lncRNA gene scriptome map of the expression and m6A modifcation expression data. Ten, the gene IDs were transformed to profles of lncRNA in GC is of great signifcance to gain gene names based on information in the Ensembl data- understanding of the mechanisms by which lncRNA base (http:// asia. ensem bl. org/ info/ data/ index. html). afects the prognosis of GC. According to the m6A-related gene type (“writers”, Immune cell score analysis improve the prognos- “readers”, “erasers”) and the corresponding gene name, tic evaluation of gastrointestinal cancer patients. m6A-related gene expression data were extracted with Immune-risk score provides prognostic evaluation and the limma package in R software (https:// www.r- proje adjuvant treatment guidance, which enhances our com- ct. org/). Next, co-expression analysis was carried out to prehension of the relationship between chemotherapy identify the correlation among m6A-related gene expres- and the immune system to broaden the claim of immu- sion and lncRNAs. According to the correlation coef- notherapy [9]. Immune scoring system improves prog- cient, we obtained regulatory hypotaxis between the two nosis prediction accuracy of TNM stage and distinguish types. By this method, we acquired the expression data GC patients with better prognosis, which might help us of m6A-related lncRNAs through the limma package in identify GC patients who would get help from adjuvant R software (http:// bioco nduct or. riken. jp/ packa ges/3. 0/ chemotherapy [10]. Te immunoscore is a measure of bioc/ html/ limma. html). Moreover, a network plot was cytotoxic immune reactions, which guide the formula- depicted via the igraph package to visualize the corre- tion of treatment strategies, and can improve the progno- lation. Expression data of m6A-related lncRNAs were sis of collision tumors [11]. Regrettably, few researchers combined with clinical survival data via the limma pack- have investigated the correlation between m6A lncRNA age. Prognostic-related lncRNAs were extracted, and and immune cell infltration in GC. Tus, it is important the confdence interval and hazard ratio were calculated to analyze immune cell infltration in the tumor micro- by the survival package. Visualization of univariate Cox environment and clarify its relationship with the clinico- regression analysis was achieved by drawing forest plots. pathological parameters of gastric cancer. Te diferential expression data of prognosis-related m6A lncRNAs between tumor and normal tissues were Materials and Methods acquired through the limma package, pheatmap pack- Sample Data Acquisition and Collation age, reshape2 package and ggpubr package in R software. We downloaded gastric cancer gene expression profles Diferences with P < 0.05 were considered statistically and clinical data from the TCGA through the Genomic signifcant. To visualize the diferences in expression, we Data Commons Data Portal [12] (https:// portal. gdc. drafted heatmaps
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