动力蛋白轻链LC8-1型在肺腺癌中的表达、作用机制和临床价值

The expression, mechanism and clinical value of dynein light chain LC8-type 1 in lung adenocarcinoma

目的探讨动力蛋白轻链LC8-1型(DYNLL1)在肺腺癌(LUAD)中的表达、作用机制和临床价值。方法本研究为实验研究。从TCGA数据库中收集539例LUAD组织和59例癌旁正常肺组织的DYNLL1 mRNA测序数据,收集GTEx数据库中的正常肺组织样本增加对照样本量(共347例正常肺组织)。收集LUAD患者的临床特征包括年龄、性别、吸烟史、TNM分期和临床分期。选取2020年1月至2022年12月于河北省胸科医院行手术切除的40例LUAD患者为研究对象,收集手术过程中切除的LUAD组织和癌旁正常肺组织用于免疫组织化学检测DYNLL1蛋白表达。通过最小P值法确定DYNLL1的高表达组和低表达组。采用Kaplan-Meier法绘制生存曲线,分析DYNLL1表达对LUAD患者总生存期(OS)、疾病特异性生存期和无进展生存期的影响。采用单因素和多因素Cox回归分析LUAD患者OS的影响因素。通过GEPIA基因表达谱动态分析数据库获取LUAD中与DYNLL1显著相关的前100个基因的表达情况,并进行基因本体论(GO)和京都基因和基因组数据库(KEGG)富集分析,进一步通过FunRich软件进行功能富集分析。应用TIMER数据库对DYNLL1进行免疫浸润分析和泛癌中的表达分析。通过TCGA数据库中LUAD的表达数据分析DYNLL1表达与甲基转移酶的关系。通过DiseaseMeth version 2.0在线数据库对DYNLL1在LUAD组织中的甲基化水平进行分析。利用MEXPRESS在线数据库分析DYNLL1相关甲基化位点。使用String数据库分析DYNLL1的蛋白质-蛋白质相互作用。结果相比正常组织,DYNLL1 mRNA在多种实体肿瘤组织中均高表达(均P<0.05)。TCGA数据库、TCGA和GTEx数据库中,LUAD组织的DYNLL1 mRNA水平均高于正常肺组织(均P<0.05)。免疫组织化学检测显示,DYNLL1主要定位于细胞质或细胞核。DYNLL1蛋白在LUAD组织中的高表达率高于癌旁正常肺组织[80.0%(32/40)比40.0%(16/40),χ^(2)=13.33,(P<0.001)]。男性、T分期为T2+T3+T4期、N分期为N1+N2+N3期、临床分期为Ⅲ期+Ⅳ期LUAD患者的DYNLL1 mRNA表达水平高于女性、T分期为T1期、N分期为N0期、临床分期为Ⅰ期+Ⅱ期患者(均P<0.01)。DYNLL1高表达组和低表达组的总体生存曲线、疾病特异性生存曲线和无进展生存曲线显示,低表达组的生存状态均优于高表达组(HR分别为1.902、1.863、1.662,均P<0.001)。多因素Cox回归分析显示DYNLL1是LUAD患者OS的影响因素(HR=1.467,95%CI:1.041~2.068,P=0.029)。GO富集分析、KEGG富集分析和FunRich软件功能富集分析显示,DYNLL1可能通过调节细胞周期调控LUAD的发生发展。DYNLL1 mRNA表达与B淋巴细胞(r=-0.261,P<0.001)、CD8^(+)T淋巴细胞(r=0.105,P=0.020)、CD4^(+)T淋巴细胞(r=-0.137,P=0.002)和树突状细胞(r=-0.178,P<0.001)具有相关性,与CD19、CD1C、CD2、CD3E、CD4、CD79A、HLA-DPA1、HLA-DPB1、HLA-DQB1、HLA-DRA、ITGAX和NRP1具有相关性。3种甲基转移酶(DNMT1、DNMT3A和DNMT3B)在高表达组和低表达组之间的差异均有统计学意义。LUAD组织中DYNLL1甲基化水平高于正常肺组织(P=0.007)。在DYNLL1的DNA序列中发现了6个甲基化位点。与DYNLL1相互作用的蛋白质包括DYNLRB1、BCL2L11、DYNC1I2、DYNC1I1、WDR34、DYNLT1、DYNC1H1、WDR60、STRN4和MOB4。结论DYNLL1在LUAD组织中高表达,可能通过调节细胞周期、参与免疫细胞浸润过程和甲基化等对LUAD的发生发展产生影响,与患者的不良预后密切相关。

Objective:To investigate the expression,mechanism,and clinical significance of dynein light chain LC8-type 1(DYNLL1)in patients with lung adenocarcinoma(LUAD).Methods:It was an experimental study.DYNLL1 mRNA sequencing data from 539 LUAD tissues and 59 adjacent normal lung tissues were available from The Cancer Genome Atlas(TCGA)database.Normal lung tissue samples were additionally collected from the Genotype-Tissue Expression(GTEx)database to increase the sample size in the control group(totally 347 normal lung tissue samples).Clinical characteristics of LUAD patients,including age,gender,smoking history,TNM stage,and clinical stage,were collected.Forty LUAD patients who underwent surgical resection in Hebei Chest Hospital from January 2020 to December 2022 were selected for the study.LUAD tissues and adjacent normal lung tissues intraoperatively collected were used for immunohistochemical detection of DYNLL1 protein expression.High and low expression groups of DYNLL1 were determined using the minimum P-value method.Kaplan-Meier survival curves were plotted to analyze the impact of DYNLL1 expression on the overall survival(OS),disease specific survival(DSS),and progression free survival(PFS)in LUAD patients.Univariate and multivariate Cox regression analyses were conducted to determine influencing factors for OS in LUAD patients.The top 100 genes significantly associated with DYNLL1 in LUAD were obtained from the Gene Expression Profiling Interactive Analysis(GEPIA)database,followed by the gene ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analyses.Functional enrichment analysis was further conducted using FunRich software.The Tumor Immunity Estimation Resource(TIMER)database was used to analyze immune infiltration and pan-cancer expression of DYNLL1 in LUAD.The correlation between DYNLL1 expression and methyltransferase was analyzed using LUAD expression data from the TCGA database.The methylation level of DYNLL1 in LUAD tissues was analyzed using the DiseaseMeth version 2.0 online database.DYNLL1-related methylation sites were analyzed using the MEXPRESS online database.Protein-protein interaction of DYNLL1 were analyzed using the String database.Results:Compared to that of normal tissues,the mRNA level of DYNLL1 was significantly highly in various solid tumor tissues(all P<0.05).In the TCGA database,the TCGA and GTEx databases,the mRNA level of DYNLL1 in LUAD tissues was significantly higher than that of normal lung tissues(all P<0.05).Immunohistochemistry showed that DYNLL1 was primarily localized in the cytoplasm or nucleus.The positive expression rate of DYNLL1 in LUAD tissues was significantly higher than that of adjacent normal lung tissues(80.0%[32/40]vs 40.0%[16/40],χ^(2)=13.33,P<0.001).The mRNA level of DYNLL1 was significantly higher in male patients,patients with T2+T3+T4 stages,N1+N2+N3 stages,and clinical stagesⅢ+Ⅳcompared to female patients,patients with T1 stage,N0 stage,and clinical stagesⅠ+Ⅱ(all P<0.01).OS,DSS and PFS curves of LUAD patients with high and low DYNLL1 levels were depicted.LUAD patients in the low expression group had a significantly better survival(OS,DSS and PFS)than the high expression group(HR=1.902,1.863,and 1.662,respectively;all P<0.001).Multivariate Cox regression analysis indicated that DYNLL1 was a significant factor affecting OS in LUAD patients(HR=1.467,95%CI:1.041-2.068,P=0.029).GO,KEGG and FunRich functional enrichment analyses suggested that DYNLL1 might regulate the occurrence and development of LUAD by modulating the cell cycle.The mRNA level of DYNLL1 was significantly correlated with B lymphocytes(r=-0.261,P<0.001),CD8^(+)T lymphocytes(r=0.105,P=0.020),CD4^(+)T lymphocytes(r=-0.137,P=0.002),and dendritic cells(r=-0.178,P<0.001),and it was correlated with CD19,CD1C,CD2,CD3E,CD4,CD79A,HLA-DPA1,HLA-DPB1,HLA-DQB1,HLA-DRA,ITGAX,NRP1.There were significant differences in the expression levels of three methyltransferases(DNMT1,DNMT3A,DNMT3B)between high and low expression groups.DYNLL1 methylation levels in LUAD tissues were significantly higher than those of normal lung tissues(P=0.007).Six methylation sites were found in the DNA sequence of DYNLL1.Proteins interacted with DYNLL1 included DYNLRB1,BCL2L11,DYNC1I2,DYNC1I1,WDR34,DYNLT1,DYNC1H1,WDR60,STRN4,MOB4.Conclusions:DYNLL1 is overexpressed in LUAD tissues and it may influence the occurrence and development of LUAD through regulating the cell cycle,immune cell infiltration,and methylation.DYNLL1 is closely related to poor prognosis in LUAD patients.