基于横纹肌肉瘤融合基因状态的EMT相关lncRNA-miRNA-mRNA调控网络  

Regulatory network of EMT-related lncRNA-miRNA-mRNA based on rhabdomyosarcoma fusion gene states

作　　者：何林蔚 孟莲 刘春霞 HE Lin-Wei;MENG Lian;LIU Chun-Xia(Department of Pathology,Shihezi University School of Medicine,Shihezi 832002,Xinjiang,China)

机构地区：[1]石河子大学医学院病理学系,新疆石河子832002 [2]石河子大学医学院第一附属医院病理科,新疆石河子832002 [3]广州医科大学附属第二医院病理科

出　　处：《中国老年学杂志》2024年第20期4967-4975,共9页Chinese Journal of Gerontology

基　　金：国家自然科学基金项目(81960485,82060487);广州市科技计划市校联合项目(202201020104)。

摘　　要：目的基于横纹肌肉瘤(RMS)融合基因阳性与阴性状态,鉴定与上皮间质转化(EMT)及免疫相关的竞争性内源RNA(ceRNA)网络,初步分析网络中关键分子富含鞘糖脂的微结构域(PAG)1和hsa-miR-30a-5p的调控关系与预后。方法从高通量基因表达(GEO)数据库获取RMS数据集GSE108022、GSE66533与GSE97553,以融合基因阳性和阴性进行分组。基因富集(GSEA)Hallmark分析后,提取差异表达基因、miRNAs和lncRNAs,进行基因本体论(GO)与京都基因与基因组百科全书(KEGG)分析。从EMT标志物泛癌分析(EMTome)数据库提取EMT相关基因并取交集。运用miRDB、Encori和DIANA数据库鉴定RNA交互关系,Cytoscape构建lncRNA-miRNA-mRNA网络,Kaplan-Meier plotter数据库进行预后分析,R语言进行免疫相关性分析。转染hsa-miR-30a-5p inhibitor后,将RMS细胞RD与RH30分为inhibitor NC与inhibitor组。运用实时荧光定量-聚合酶链反应(qRT-PCR)与Western印迹检测PAG1和hsa-miR-30a-5p在RMS细胞中的表达及调控关系;Western印迹检测EMT相关标志物蛋白表达;平板克隆实验、Transwell迁移和侵袭实验验证hsa-miR-30a-5p对RMS细胞功能的影响。结果GSEA结果表明,数据集基因主要富集在肿瘤EMT通路;GO和KEGG结果表明,差异表达基因和miRNAs的功能主要富集于与肿瘤迁移、侵袭相关的通路(P<0.05);PAG1表达越低、hsa-miR-30a-5p表达越高的患者预后越差(P<0.05);在融合阳性样本中,PAG1表达水平与浆细胞样树突细胞和1型辅助性T细胞呈正相关(P<0.05)。PAG1在融合阳性的RMS细胞中低表达,hsa-miR-30a-5p高表达;与NC组相比,inhibitor组PAG1 mRNA与蛋白表达量明显上升(P<0.05),E-钙黏蛋白表达明显升高,N-钙黏蛋白与波形蛋白表达明显降低(P<0.05);在RH30细胞中,与NC组相比,inhibitor组RMS细胞的增殖、迁移及侵袭能力明显下降(P<0.05)。结论基于RMS融合基因状态构建与EMT及免疫相关lncRNA-miRNA-mRNA调控网络,筛选出与RMS免疫及预后相关的Objective Based on the fusion positive and negative gene status of rhabdomyosarcoma(RMS),the competing en-dogenous RNA(ceRNA)network related to epithelial-mesenchymal transition(EMT)and immunity was identified,and the regulatory relationship and prognostic significance of key molecules phosphoprotein associated with glycosphingolipid-enriched microdomains(PAG)1 and hsa-miR-30a-5p in the network were preliminarily analyzed.Methods RMS data sets GSE108022,GSE66533 and GSE97553 were obtained from Gene Expression Omnibus(GEO)database and grouped according to fusion-positive genes and negative genes.After gene enrichment(GSEA)Hallmark analysis,differentially expressed genes,miRNAs and lncRNAs were extracted and an-alyzed by gene ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG).EMT related genes were extracted from pan-cancer analysis of EMT markers(EMTome)database and intersected with the differentially expressed genes.miRDB,Encori and DI-ANA databases were used to identify RNA interaction,Cytoscape was used to construct lncRNA-miRNA-mRNA network,Kaplan-Meier plotter database was used for prognosis analysis,and R language was used for immune correlation analysis.After transfection with hsa-miR-30a-5p inhibitor,RMS cell lines RD and RH30 were divided into inhibitor NC and inhibitor groups.Real-time fluorescence quan-tification-polymerase chain reaction(qRT-PCR)and Western blot were used to detect the expressions and regulatory relationship of PAG1 and hsa-miR-30a-5p in RMS cell lines;Western blot was used to detect protein expressions in EMT related biomarkers;the effects of hsa-miR-30a-5p on the function of RMS cells were validated through plate cloning experiment,Transwell migration and inva-sion assays.Results GSEA results showed that the genes of the datasets were mainly enriched in tumor EMT pathway;GO and KEGG results showed that the functions of differentially expressed genes and miRNAs were mainly enriched in the pathways related to tumor invasion and migration(P<0.05);patients with lower expressio

关 键 词：融合基因 横纹肌肉瘤 上皮间质转化 竞争性内源RNA网络 免疫 

分 类 号：R738.6[医药卫生—肿瘤]