基于生物信息学方法分析长链非编码RNA LINC00494在肺腺癌中的表达及诊疗与预后价值

Expression level of long non-coding RNA LINC00494 in lung adenocarcinomas and its diagnostic and prognostic values based on bioinformatics methods

目的探讨LINC00494在肺腺癌中的表达及诊疗与预后价值。方法本研究为实验研究。采用生物信息学分析方法,检索TCGA数据库中535个肺腺癌患者组织和59个正常肺组织的RNA-seq数据,以及相应的患者临床资料(生存信息、性别、年龄、肿瘤长径、临床分期和淋巴结转移情况)。根据LINC00494在肺腺癌患者组织表达的中位数,分为高表达组(224例)和低表达组(224例),比较2组患者临床特征。采用Log-rank检验与多因素Cox回归分析明确LINC00494与肺腺癌患者预后的关系。采用lncATLAS在线数据库分析LINC00494的亚细胞定位情况。通过共表达分析方法获得与LINC00494共表达的蛋白质编码基因,对其进行基因本体论(GO)和京都基因和基因组百科全书(KEGG)富集分析、蛋白质-蛋白质相互作用网络分析,探讨LINC00494在肺腺癌中的功能。采用非随机抽样的方法收集2016年1月至2020年12月于西安交通大学第二附属医院经病理学检查确诊为肺腺癌的21例患者,通过将21对肺腺癌及其对应癌旁组织标本进行实时荧光定量PCR(RT-qPCR)实验,验证LIN00494在肺腺癌组织中的表达情况。采用3种肺腺癌细胞系A549、H1299和PC-9,以及正常支气管上皮细胞系BEAS-2B进行RT-qPCR,验证LINC00494的表达水平。将H1299细胞进行细胞核与细胞质分离,确定LINC00494的亚细胞定位。结果LINC00494在肺腺癌组织中较正常肺组织中表达上调。高表达组和低表达组患者的性别、肿瘤长径和临床分期比较差异均有统计学意义(均P<0.01),年龄和淋巴结转移比较差异均无统计学意义(均P>0.05)。生存分析结果显示高表达组的生存状态优于低表达组(χ^(2)=7.24,P=0.007),高表达组的总生存期和中位总生存期均长于低表达组[(7.14±1.82)年比(5.36±1.62)年、(3.89±1.29)年比(3.27±0.87)年]。多因素Cox回归分析结果显示临床分期Ⅱ期、Ⅲ期、Ⅳ期是影响肺腺癌患者预后的独立危险因素,LINC00494高表达水平是影响肺腺癌患者预后的独立保护因素(均P<0.05)。lncATLAS在线数据库分析发现LINC00494定位于细胞核。通过共表达分析方法,获得247个与LINC00494共表达的蛋白质编码基因。GO分析显示与LINC00494共表达的蛋白质编码基因主要富集通路为信号转导、免疫应答、炎性反应、免疫应答调节、细胞表面受体信号等。KEGG分析显示与LINC00494共表达的蛋白质编码基因主要富集通路为细胞因子受体间相互作用、原发性免疫不全、细胞黏附分子、EB病毒感染、T细胞受体信号通路等。21例患者中男13例,女8例;年龄(60.57±10.41)岁,年龄范围40~76岁;肺腺癌组织LINC00494表达较癌旁组织高,差异有统计学意义[(1.954±0.075)比(0.986±0.056),t=2.73,P=0.013]。4种细胞系的LINC00494表达水平差异有统计学意义(F=109.06,P<0.001)。H1299和PC-9肺腺癌细胞系的LINC00494表达水平较正常支气管上皮细胞系BEAS-2B升高[(4.860±0.603)比(1.959±0.168)比(1.002±0.008)],差异均有统计学意义(均P<0.05)。在H1299细胞中,LINC00494约有87%在细胞核中表达。结论LINC00494在肺腺癌中表达上调,且与肺腺癌预后良好有关。LINC00494定位于细胞核,参与多条免疫反应相关通路,是潜在的肺腺癌诊断、治疗和预后评估的生物标志物。

Objective To investigate the expression level of long non-coding RNA LINC00494 in lung adenocarcinomas(LUADs)and its diagnostic and prognostic values.Methods This was an experimental study.RNA-seq data of 535 LUAD tissues and 59 normal lung tissues,and corresponding clinical data(survival data,sex,age,tumor diameter,TNM staging,lymph node metastasis)were collected from the TCGA database using bioinformatic methods.According to the median expression of LINC00494 in LUADs,patients were assigned into high expression group(n=224)and low expression group(n=224).Clinical characteristics were compared between groups.The correlation between expression level of LINC00494 and prognosis of LUAD patients was analyzed by Log-rank test and multivariate Cox regression analysis.The subcellular localization of LINC00494 was analyzed using the LncATLAS online database.The protein coding genes co-expressed with LINC00494 were obtained by co-expression analysis.GO and KEGG enrichment analyses and protein-protein interaction analysis were performed to explore the function of LINC00494 in LUAD.A total of 21 LUAD patients pathologically diagnosed in the Second Affiliated Hospital of Xi′an Jiaotong University from January 2016 to December 2020 were randomly selected by the non-random sampling method.Real-time quantitative PCR(RT-qPCR)was performed to verify the expression level of LIN00494 in 21 LUAD tissues and paracancerous tissues.Its expression in three LUAD cell lines A549,H1299 and PC-9 was measured by RT-qPCR.Subcellular localization of LINC00494 in nuclear and cytoplasmic H1299 cells was identified.Results LINC00494 was highly expressed in LUAD tissues compared with that of normal tissues.There were significant differences in the sex,tumor diameter and TNM staging between high expression group and low expression group(P<0.01),but no significant differences in the age and lymph node metastasis were detected between groups(P>0.05).Survival analysis showed that the survival state of the high expression group was significantly better than that of the low expression group(χ^(2)=7.24,P=0.007),presenting longer overall survival(OS,[7.14±1.82]years vs[5.36±1.62]years)and median OS([3.89±1.29]years vs[3.27±0.87]years).Multivariate Cox regression analysis showed that stageⅡ,ⅢandⅣwere independent risk factors for the prognosis of LUAD,and high expression level of LINC00494 was an independent protective factor(all P<0.05).LINC00494 was localized in the nucleus as analyzed by LncATLAS online database.Through co-expression analysis,247 protein-coding genes that co-expressed with LINC00494 were identified.GO analysis showed that protein-coding genes co-expressed with LINC00494 were mainly enriched in signal transduction,immune response,inflammatory response,regulation of immune response and cell surface receptor signaling pathway.Also,KEGG enrichment analysis showed that these protein-coding genes were mainly enriched in cytokine-cytokine receptor interaction,primary immunodeficiency,cell adhesion molecule,Epstein-Barr virus infection,and T cell receptor signaling pathway.Among the 21 LUAD patients,there were 13 male and 8 female.Their mean age was 60.57±10.41(40-76)years.LINC00494 was significantly up-regulated in LUAD tissues than that of paracancerous tissues([1.954±0.075]vs[0.986±0.056],t=2.73,P=0.013).A significant difference in the expression level of LINC00494 was detected among the four cell lines(F=109.06,P<0.001).LINC00494 was significantly highly expressed in LUAD cell lines H1299 and PC-9 than that of the normal bronchial epithelial cell line BEAS-2B([4.860±0.603]vs[1.959±0.168]vs[1.002±0.008],P<0.05).In H1299 cells,approximately 87%of LINC00494 was expressed in the nucleus.Conclusions LINC00494 is up-regulated in LUAD and associated with a good prognosis.LINC00494 is localized in the nucleus and participates in immune response-related pathways.It is a promising biomarker for the diagnosis,treatment and prognostic evaluation of LUAD.