Decreased zinc-fingers and homeoboxes family expression was associated with unfavorable outcomes and immune infiltration in lung adenocarcinoma

Abstract

Background: The zinc-fingers and homeoboxes (ZHX) family is a group of nuclear homodimeric transcriptional repressors that play an essential role in developing and progressing diverse malignancies. However, the association of ZHX family expression with prognosis and immune infiltration in lung adenocarcinoma (LUAD) is still unclear. The current study aimed to investigate the relationship between ZHX family expression and clinical outcomes and immune infiltration in LUAD patients.

Methods: ZHXs family expression was determined by using the Oncomine database and Cancer Cell Line Encyclopedia (CCLE). The impact of ZHXs family expression on prognosis was analyzed by using the Kaplan-Meier-plotter online database. The Search Tool for the Retrieval of Interacting Genes (STRING) database was utilized to construct the interaction network based on the selected differentially expressed genes associated with ZHXs. The Database for Annotation, Visualization, and Integrated Discovery (DAVID) was used to perform the enrichment of the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. The functional state of the ZHXs family in diverse types of malignancies was determined by CancerSEA. The Tumor Immune Estimation Resource (TIMER) database was used to evaluate the association of the ZHXs family with immune cell infiltrates. ZHXs family expression was validated by the Gene Expression Omnibus (GEO) database and real-time polymerase chain reaction (RT-PCR) in 10 paired tumors and normal tissues.

Results: ZHX1-3 expression level significantly decreased in LUAD compared with normal tissues. Attenuated ZHXs expression was significantly associated with unfavorable overall survival in LUAD patients. ZHX family members were positively associated with immune infiltration of monocytes, tumor-associated macrophages (TAMs), M1 and M2 macrophages in LUAD. ZHX family expression was also significantly related to a variety of immune marker sets in LUAD. GEO analysis and RT-PCR validated the significant decrease of ZHXs expression level in LUAD.

Conclusions: The current study revealed that ZHX family expression was significantly correlated with unfavorable outcomes and immune infiltration in LUAD. The findings herein provide a promising basis for further study into the potential biological function of the ZHX family in LUAD and lay a foundation for developing therapeutic targets for LUAD patients.

Keywords: Zinc-fingers and homeoboxes family (ZHX family); immune infiltration; lung adenocarcinoma (LUAD); prognosis.

Figure 1

ZHX family expression levels in various types of human cancers. (A) Different levels of ZHX family members’ expression in datasets of various malignancies in the Oncomine database. (B-D) Expression levels of ZHX family members in various malignancies analyzed by TIMER (*, P<0.05; **, P<0.01; ***, P<0.001). ZHX, zinc-fingers and homeoboxes; TPM, transcripts per million; TIMER, tumor immune Estimation Resource.

Figure 2

Overall survival analysis in Kaplan-Meier plotter database for mRNA levels of ZHX factors in LUAD patients. (A-C) The overall survival curves of ZHX1, ZHX2 and ZHX3 in LUAD patients, respectively. (D-F) The overall survival curves of ZHX1, ZHX2, and ZHX3 in LUSC patients, respectively. ZHX, zinc-fingers and homeoboxes; HR, hazard ratio; LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma.

Figure 3

GO annotation and KEGG pathways enrichment analysis for ZHX family members and relevant genes. (A) The interaction network among the selected DEGs that are most relevant to ZHXs was analyzed by the STRING database. (B) BP, CC, and MF were the three main categories. The y-axis represents the enrichment score in each category, and the x-axis represents different GO terms. (C) Scatter plot of enriched KEGG pathways statistics. The top 10 KEGG enriched pathways are shown in the figure. ZHX, zinc-fingers and homeoboxes; BP, biological process; CC, cellular component; MF, molecular function; ATP, adenosine triphosphate; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; DEGs, differentially expressed genes; STRING, Search Tool for the Retrieval of Interacting Genes.

Figure 4

The functional state of ZHX family in fifteen types of cancer. (A-C) Represented the functional state of ZHX1, ZHX2, and ZHX3, respectively. The blue plots represent that ZHX family member expression was negatively correlated with the functional state, while the red plots represent that ZHX family member expression was positively correlated with the functional state determined by CancerSEA. EMT, epithelial-mesenchymal transition; CNS, central nervous system; AML, acute myeloid leukemia; ALL, acute lymphocytic leukemia; CML, chronic myelogenous leukemia; GBM, glioblastoma; AST, astrocytoma; HGG, high grade glioma; ODG, oligodendroglioma; LUAD, lung adenocarcinoma; NSCLC, non-small-cell lung cancer; MEL, melanoma; RCC, renal cell carcinoma; BRCA, breast cancer; PC, prostate cancer; HNSCC, head and neck squamous cell carcinoma; CRC, colorectal cancer; RB, retinoblastoma; UM, uveal melanoma; OV, ovarian cancer; ZHX, zinc-fingers and homeoboxes.

Figure 5

Correlation of expression of ZHX family member with immune infiltration levels in LUAD. (A) Expression of ZHX1 showed a significant positive correlation with infiltrating levels of dendritic cells, CD4⁺ T cells, CD8⁺ T cell, macrophages, and neutrophils; no significant correlations with tumor purity and infiltrating levels of B cell. (B) ZHX2 was significantly associated with infiltrating levels of dendritic cells, neutrophils, CD4⁺ T cells, CD8⁺ T cell, B cell and macrophages. (C) ZHX3 expression was significantly corelated with infiltrating levels of dendritic cells, neutrophils, macrophages and CD4⁺ T cell; no significant correlation was observed with tumor purity and infiltrating levels of CD8⁺ T cell and B cell. Cor, correlation; ZHX, zinc-fingers and homeoboxes; TPM, transcripts per million; LUAD, lung adenocarcinoma.

Figure 6

The validation of ZHX family member expression in LUAD and normal tissues by GEO database. (A,D,H) Indicated that the ZHX1 was significantly decreased in 354 patients of LUAD compared to 168 controls in the three datasets (GSE19188, GSE31210, and GSE75037). (B,E,I) Indicated that the ZHX2 was also significantly decreased in LUAD compared to normal tissue. (C,F,G) Showed that the ZHX3 was significantly lower in LUAD compared to normal tissue. The ZHX family member expression was analyzed using an unpaired t-test. P<0.05 were considered to be statistically significant (***, P<0.001). ZHX, zinc-fingers and homeoboxes; LUAD, lung adenocarcinoma; GEO, Gene Expression Omnibus.

Figure 7

The validation of ZHX family member expression in LUAD and normal tissues by RT-PCR in 10 pairs of LUAD and normal tissues. (A-C) Indicated that ZHX1, ZHX2, and ZHX3 expression levels were significantly lower in LUAD than in normal tissues. ZHX, zinc-fingers and homeoboxes; LUAD, lung adenocarcinoma; RT-PCR, real-time polymerase chain reaction.