基于生物信息学分析探讨肝癌BRD4与预后关系及其ceRNA调控网络构建

doi:10.3877/cma.j.issn.2095-3232.2024.04.022

目的

基于生物信息分析探讨肝细胞癌（HCC）溴结构域蛋白4（BRD4）与预后关系及其竞争性内源性RNA（ceRNA）调控网络的构建。

方法

TCGA数据库中将HCC样本分为两组，采用生存R软件包分析评估BRD4对总体生存期（OS）的影响。TIMER数据库评估免疫细胞的浸润水平，采用Spearman相关分析法分析BRD4表达与癌症免疫浸润水平的相关性。采用ClusterProfiler R软件包对KEGG进行基因集富集分析。从癌症基因组图谱、基因型-组织表达和GSE147889数据集收集了HCC及正常对照的lncRNAs、microRNAs（miRNAs，miR）和mRNAs表达数据并进行差异分析，预测调控其表达的miRNAs及lncRNAs，构建ceRNA调控网络。从新疆维吾尔自治区人民医院收集了10个HCC组织样本及匹配的非癌症组织样本，检测BRD4及相关ceRNA网络基因的表达。在HCCLM3细胞中进行过表达和敲降BRD4，进行凋亡、侵袭和伤口愈合试验。

结果

生存分析显示BRD4高表达的HCC患者生存率较差（HR=1.020，P<0.05）。BRD4的高表达与B细胞、CD4⁺ T细胞、中性粒细胞、巨噬细胞和树突细胞呈正相关（r_s=0.265，0.433，0.302，0.332，0.258；P<0.05）。基因集富集分析显示，BRD4可能与胰岛素信号通路、抑制氧化磷酸化有关。通过3个数据库差异分析获得2个DEmRs调控因子miR-200a-3p和miR-141-3p，鉴定出2个交集lncRNAs（MYLK-AS1和DLX6-AS1），并构建ceRNA网络。组织样本检测证实BRD4在HCC中的表达高于对照组。敲除BRD4显著抑制HCCLM3细胞的侵袭和迁移，并促进细胞凋亡。双荧光素酶测定证实miR-200a-3p调节的BRD4和MYLK-AS1、DLX6-AS1是上游海绵。

结论

BRD4在HCC中的高表达与不良预后相关，BRD4过表达促进了HCC细胞的侵袭和迁移，并通过ceRNA调控机制参与HCC发生和发展。

关键词: 癌，肝细胞, 溴结构域蛋白4（BRD4）, 竞争性内源性RNA （ceRNA）, 免疫细胞浸润

Objective

To investigate the relationship between BRD4 and prognosis of hepatocellular carcinoma (HCC) based on bioinformatics analysis and the construction of competitive endogenous RNA (ceRNA) regulatory network.

Methods

HCC samples from TCGA database were divided into two groups. The effect of BRD4 on overall survival (OS) was evaluated by R software package. The infiltration level of immune cells was evaluated by TIMER database. The correlation between BRD4 expression and cancer immune infiltration level was analyzed by Spearman's correlation analysis. KEGG gene set enrichment analyses was performed by ClusterProfiler R software package. The expression levels of lncRNAs, microRNAs (miRNAs, miR) and mRNAs in HCC and normal controls were collected from TCGA, Genotype-Tissue Expression (GTEx) and GSE147889 datasets, and the differences were analyzed. The miRNAs and lncRNAs regulating their expression profiles were predicted and a ceRNA regulatory network was constructed. 10 HCC tissue samples and matched non-cancer tissue samples were collected from People's Hospital of Xinjiang Uygur Autonomous Region. The expression levels of BRD4 and related ceRNA network genes were detected. BRD4 was overexpressed and knocked down in HCCLM3 cells. Cell apoptosis, invasion and wound healing assays were carried out.

Results

Survival analysis showed that HCC patients with high BRD4 expression obtained poor survival rate (HR=1.020, P<0.05). High expression of BRD4 was positively correlated with B cells, CD4⁺ T cells, neutrophils, macrophages and dendritic cells (r_s=0.265, 0.433, 0.302, 0.332, 0.258; P<0.05). Gene set enrichment analysis showed that BRD4 was probably correlated with insulin signaling pathway and inhibition of oxidative phosphorylation. 2 DEmRs regulatory factors of miR-200a-3p and miR-141-3p were obtained through the analysis of differences among 3 databases, and 2 overlapping lncRNAs (MYLK-AS1 and DLX6-AS1) were identified, and the ceRNA network was constructed. Tissue sample detection confirmed that the expression of BRD4 in HCC was higher than that in normal controls. Knockout of BRD4 significantly inhibited the invasion and migration and promoted cell apoptosis of HCCLM3 cells. Dual luciferase assay confirmed that BRD4, MYLK-AS1 and DLX6-AS1 regulated by miR-200a-3p were the upstream sponges.

Conclusions

High expression of BRD4 in HCC is associated with poor prognosis. Overexpression of BRD4 promotes the invasion and migration of HCC cells, and participates in the incidence and development of HCC through ceRNA regulatary mechanism.

Key words: Carcinoma, hepatocellular, Bromodomain-containing protein (BRD4), Competing endogenous RNA (ceRNAs), Immune cell infiltration

表1 本研究所用引物

基因	序列
miR-200a-3p	F: 5′-TAACACTGTCTGGTAACGATGT-3′
	R: 5′-CATCTTACCGGACAGTGCTGGAT-3′
miR-141-3p	F: 5′-CGGGCTAACACTGTCTGGTAAAG-3′
	R: 5′-GTGCAGGGTCCGAGGTATTC-3′
U6	F: 5′-CTCGCTTCGGCAGCACA-3′
	R: 5′-AACGCTTCACGAATTTGCGT-3′
BRD4	F: 5′-GGACTAGAAACCTCCCAAATGTCTACAA-3′
	R: 5′-CCTGTGTGCACTTGCTCCCGAGGAGAGA-3′
MYLK-AS1	F: 5′-TTGCAGTGTTCAGCACTGGCAC-3′
	R: 5′-ATTCGACGACCAGTGTTTCAGT-3′
DLX6-AS1	F: 5′-AGTTTCTCTCTAGATT-GCCTT-3′
	R: 5′-ATTGACATGTTAGTGCCCTT-3′
GAPDH	F: 5′-TGTGGGCATCAATGGATTTGG-3′
	R: 5′-ACACCATGTATTCCGGGTCAAT-3′

表1 本研究所用引物

基因	序列
miR-200a-3p	F: 5′-TAACACTGTCTGGTAACGATGT-3′
	R: 5′-CATCTTACCGGACAGTGCTGGAT-3′
miR-141-3p	F: 5′-CGGGCTAACACTGTCTGGTAAAG-3′
	R: 5′-GTGCAGGGTCCGAGGTATTC-3′
U6	F: 5′-CTCGCTTCGGCAGCACA-3′
	R: 5′-AACGCTTCACGAATTTGCGT-3′
BRD4	F: 5′-GGACTAGAAACCTCCCAAATGTCTACAA-3′
	R: 5′-CCTGTGTGCACTTGCTCCCGAGGAGAGA-3′
MYLK-AS1	F: 5′-TTGCAGTGTTCAGCACTGGCAC-3′
	R: 5′-ATTCGACGACCAGTGTTTCAGT-3′
DLX6-AS1	F: 5′-AGTTTCTCTCTAGATT-GCCTT-3′
	R: 5′-ATTGACATGTTAGTGCCCTT-3′
GAPDH	F: 5′-TGTGGGCATCAATGGATTTGG-3′
	R: 5′-ACACCATGTATTCCGGGTCAAT-3′

图1 BRD4与HCC患者预后和免疫浸润的关系注：a为TCGA队列中BRD4高表达和BRD4低表达患者OS的Kaplan-Meier曲线比较，b为BRD4表达与免疫细胞评分的相关性；BRD4为结构域蛋白4，TCGA为癌症基因组图谱，OS为总体生存率，HCC为肝细胞癌

图2 BRD4高表达和低表达HCC的基因GSEA分析注：a为BRD4高表达HCC样本中基因的KEGG途径分析，b为BRD4低表达HCC样本中基因的KEGG途径；BRD4为结构域蛋白4，GSEA为基因集富集分析，HCC为肝细胞癌

图3 调节BRD4的lncRNA鉴定与ceRNA网络构建注：a为Targetscan数据库、Starbase数据库和miRDB数据库中预测BRD4的miRNA调控因子；b为HCC和对照组之间差异表达的miRNAs的热图；c差异表达lncRNAs的火山图；d为BRD4的ceRNA调控网络的桑基图；BRD4为结构域蛋白4，miRNA为微核糖核酸，HCC为肝细胞癌，lncRNA为长链非编码RNA，ceRNA为竞争性内源性RNA

图4 HCC样本中BRD4的表达水平注：a为HCC和对照中BRD4的mRNA水平，***为P<0.001；b为Western blot检测HCC和对照中BRD4的蛋白质水平，***为P<0.001；NC为对照组，BRD4为结构域蛋白4，HCC为肝细胞癌

图5 BRD4影响HCC的细胞侵袭和划痕实验注：a为siRNA-BRD4 mRNA水平降低，OE-BRD4组mRNA水平升高；b、c分别为Transwell侵袭和划痕实验示siRNA-BRD4组侵袭和迁移能力减弱，OE-BRD4组增强；d为显微镜下Transwell侵袭试验细胞穿膜情况；e为流式细胞检测细胞凋亡（第四象限）；*为与NC组比较P<0.05，**为与NC组比较P<0.01，NC为对照组；BRD4为结构域蛋白4，HCC为肝细胞癌

图6 双荧光素酶测定BRD4和miR-200a-3p的靶向结合注：***为与NC组比较P<0.001；BRD4为结构域蛋白4；BRD4-mut为miR-200潜在结合位点突变，BRD4-WT为野生型BRD4

图7 qRT-PCR检测HCC的BRD4调控中差异表达基因注：***为与NC组比较P<0.001；RD4为结构域蛋白4，HCC为肝癌细胞，NC为对照组

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Relationship between BRD4 and prognosis of hepatocellular carcinoma patients based on bioinformatics analysis and construction of ceRNA regulatory network

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Relationship between BRD4 and prognosis of hepatocellular carcinoma patients based on bioinformatics analysis and construction of ceRNA regulatory network