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

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

Abstract

Abstract:

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

Jie Zhang, Guanglei Tian, Xiong Chen. Relationship between BRD4 and prognosis of hepatocellular carcinoma patients based on bioinformatics analysis and construction of ceRNA regulatory network[J]. Chinese Journal of Hepatic Surgery(Electronic Edition), 2024, 13(04): 568-576.

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References 36

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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′

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′

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