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. 2022 Mar 10:10:e12944.
doi: 10.7717/peerj.12944. eCollection 2022.

Expression of IER3 in hepatocellular carcinoma: clinicopathology, prognosis, and potential regulatory pathways

Fei-Yan He  1 , Gang Chen  1 , Rong-Quan He  2 , Zhi-Guang Huang  1 , Jian-Di Li  1 , Wei-Zi Wu  3 , Ji-Tian Chen  3 , Yu-Lu Tang  1 , Dong-Ming Li  1 , Shang-Ling Pan  4 , Zhen-Bo Feng  1 , Yi-Wu Dang  1
Affiliations

Expression of IER3 in hepatocellular carcinoma: clinicopathology, prognosis, and potential regulatory pathways

Fei-Yan He et al. PeerJ. .

Abstract

Background: Immediate early response 3 (IER3) is correlated to the prognosis of several cancers, but the precise mechanisms underlying the regulation by IER3 of the occurrence and development of hepatocellular carcinoma (HCC) remain unknown.

Methods: The expression level of IER3 was examined by using in-house immunohistochemistry (IHC), public gene chip, and public RNA-sequencing (RNA-seq). The standardized mean difference (SMD) was calculated to compare the expression levels of IER3 between HCC patients and controls. The summary receiver operating characteristics (sROC) was plotted to comprehensively understand the discriminatory capability of IER3 between HCC and non-HCC group. The Kaplan-Meier curves and the combined hazard ratios (HRs) were used to determine the prognostic value of IER3 in HCC. Moreover, differentially expressed genes (DEGs) and co-expression genes (CEGs) were used to explored the molecular mechanisms of IER3 underlying HCC. hTFtarget was used to predict the transcription factors (TFs) of IER3. The binding site of TFs and the IER3 promoter region was forecasted using the JASPAR website. The relevant ChIP-seq data were used to determine whether TF peaks were present in the IER3 transcription initiation.

Results: A significantly increased expression of IER3 protein was found in HCC tissue relative to non-HCC tissue as detected by IHC (p < 0.001). Compared to 1,263 cases of non-HCC tissues, IER3 in 1483 cases of HCC tissues was upregulated (SMD = 0.42, 95% confidence interval [CI] [0.09-0.76]). The sROC showed that IER3 had a certain ability at differentiating HCC tissues (area under the curve (AUC) = 0.65, 95% CI [0.61-0.69]). Comprehensive analysis of the effect of IER3 on the prognosis of patients with HCC demonstrated that higher IER3 expression was associated with poor prognosis in HCC (HRs = 1.30, 95% CI [1.03-1.64]). Pathway enrichment analysis revealed that IER3-related genes were mostly enriched in the PI3K-Akt signaling pathway, cancer-related signaling pathways, the p53 signaling pathway, and other signaling pathways. Regulatory factor X5 (RFX5) was identified as a possible regulator of IER3-related TF.

Conclusion: IER3 may be a potential prognostic marker for HCC. The molecular mechanisms of IER3 in HCC warrant further study.

Keywords: Gene chip; Hazard ratios (HRs); Hepatocellular carcinoma (HCC); Immediate early response 3 (IER3); Immunohistochemistry; RNA-sequencing.

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Conflict of interest statement

Gang Chen is an Academic Editor for PeerJ.

Figures

Figure 1
Figure 1. IER3 protein expression levels in HCC.
(A) Normal liver tissue sample #1 for IER3, IHC (×100, ×200, ×400), IHC score = 0. (B) Normal liver tissue sample #2 for IER3, IHC (×100, ×200, ×400), IHC score =2. (C) HCC tissue sample #1 for IER3, IHC (×100, ×200, ×400), IHC score =12. (D) HCC tissue sample #2 for IER3, IHC (×100, ×200, ×400), IHC score = 12. (E) IHC score analysis of IER3 in HCC samples and normal tissue samples. HCC, hepatocellular carcinoma; IER3, immediate early response 3; IHC, immunohistochemistry.
Figure 2
Figure 2. Prognostic values of IER3 in assessed by HCC.
(A) In-house IHC. (B) TCGA database. (C) Overall survival HR was calculated based on five data sets. HCC, hepatocellular carcinoma; IHC, immunohistochemistry; TCGA, The Cancer Genome Atlas; HR, hazard ratio.
Figure 3
Figure 3. Comprehensive expression of IER3 in HCC calculated with SMD.
(A) SMD forest map. (B) Egger’s test. (C) sensitivity analysis. HCC, hepatocellular carcinoma; SMD, standardized mean difference.
Figure 4
Figure 4. The comprehensive performance of IER3 to distinguish HCC from non-HCC.
(A) sROC. (B) Sensitivity and specific forest map. (C) DLR Positive and DLR Negative forest map. HCC, hepatocellular carcinoma; IER3, immediate early response 3; sROC, summary receiver operating characteristics; DLR, diagnostic likelihood ratio.
Figure 5
Figure 5. The Kaplan–Meier curves of HCC.
(A) DFS curve in GEPIA. (B) DFS curve in Kaplan–Meier Plotter. (C) PFS curve in Kaplan–Meier Plotter. (D) RFS curve in Kaplan–Meier Plotter. HCC, hepatocellular carcinoma. DFS, disease-free survival; PFS, progression free survival; RFS, relapse free survival.
Figure 6
Figure 6. Genetic alteration landscapes in HCC tissues.
(A) OncoPrint of IER3 alterations in HCC. (B) The overall survival of IER3 genetic alteration group and non-genetic alteration group. (C) The disease-free survival of IER3 mutant group and non-mutation group. HCC, hepatocellular carcinoma; IER3, immediate early response 3.
Figure 7
Figure 7. Enrichment analysis of the upregulated crossover genes of IER3 in HCC.
(A) Intersection Venn diagram of IER3 upregulated CEGs and DEGs. (B) Biological process. (C) Cell component. (D) Molecular function. (E) KEGG pathways. HCC, hepatocellular carcinoma; IER3, immediate early response 3; CEGs, differential expression genes; DEGs, co-expression genes; KEGG, Kyoto Encyclopedia of Genes and Genomes.
Figure 8
Figure 8. GO analyze based on GSEA analyze in HCC.
(A) The overall view in GO analyze; (B) mitotic sister chromatid segregation; (C) cell cycle checkpoint signaling; (D) microtubule cytoskeleton organization. HCC, hepatocellular carcinoma; GSEA, Gene Set Enrichment Analysis; GO, Gene Ontology.
Figure 9
Figure 9. KEGG analyze based on GSEA analyze in HCC.
(A) The overall view in KEGG analyze; (B) retinol metabolism; (C) cell cycle; (D) metabolic pathways. HCC, hepatocellular carcinoma; GSEA, Gene Set Enrichment Analysis; KEGG, Kyoto Encyclopedia of Genes and Genomes.
Figure 10
Figure 10. RFX5 activates IER3 transcription by binding to the IER3 promoter region.
(A) Intersection Venn diagram of IER3-associated upregulated genes and related TFs. (B) and (C) The binding site of RFX5 in the promoter region of IER3 and the first five binding sequences. (D) IER3 had binding peaks of RFX5 in the initial region of translation. IER3, immediate early response 3; TF, transcription factor. RFX5, Regulatory factor X5.
Figure 11
Figure 11. Comprehensive expression of RFX5 in HCC calculated with SMD.
Compared to non-HCC tissues, RFX5 mRNA in HCC tissues was upregulated. HCC, hepatocellular carcinoma; SMD, standardized mean difference.
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