Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2020 Oct 23:7:562441.
doi: 10.3389/fmolb.2020.562441. eCollection 2020.

Novel Molecular Subtypes Associated With 5mC Methylation and Their Role in Hepatocellular Carcinoma Immunotherapy

Zhuomao Mo  1 , Zhirui Cao  1 , Shaoju Luo  1 , Yan Chen  2 , Shijun Zhang  1
Affiliations
Free PMC article

Novel Molecular Subtypes Associated With 5mC Methylation and Their Role in Hepatocellular Carcinoma Immunotherapy

Zhuomao Mo et al. Front Mol Biosci. .
Free PMC article

Abstract

Background: 5-methylcytosine (5mC) has been reported in the prognosis of a variety of cancers, however, its role in hepatocellular carcinoma (HCC) has not been investigated yet. This study aimed at identifying the molecular subtypes associated with 5mC and establishing a relevant score to predict its prognosis in HCC.

Methods: Somatic gene mutation data and gene expression data were retrieved from The Cancer Genome Atlas database. Molecular subtypes were identified by unsupervised clustering based on the expression of 5mC regulators, and the molecular features of each subtype were investigated by survival, mutation, gene set variation, and immune cell infiltration analyses. Next, we performed a differentially expressed analysis based on the new subtypes and selected the overlapping genes for further analysis. We undertook univariate Cox analysis to analyze these genes and constructed a prognostic model by lasso regression analysis. Meanwhile, survival and gene set enrichment analyses were used to explore the prognosis and the relevant pathways, respectively. The LIRI cohort from the International Cancer Genome Consortium database was used as a reference to validate the 5mC subtypes and 5mC score.

Results: Twenty-one types of 5mC regulators were employed in this study, and three 5mC-associated molecular subtypes were identified. These three subtypes presented significant differences in prognosis, immune cell infiltration, immune checkpoint inhibitors, signaling pathways, and mutational features. Compared with cluster 3, cluster 2 exhibited significantly increased expression of PD-L1, TIM3, Galectin9, CTLA4, and CD80, while PD-L1, TIM3, and CD80 were higher in cluster 2 than in cluster 1. Furthermore, a 5mC-related score, composed of seven genes (SGPP2, SALL4, B3GNT7, ROR1, MYBL2, SLC7A1, and CAND2), was proven to be significantly associated with prognosis. The established subtypes and scores were thus successfully verified by the validated cohort.

Conclusion: To the best of our knowledge, this is the first study to identify a novel molecular subtype based on 5mC regulators. The identification of the 5mC-associated subtype may help reveal the potential relation between 5mC and immunity and provide novel insights for the development of individualized therapy for HCC.

Keywords: 5mC methylation; hepatocellular carcinoma; immunotherapy; molecular subtype; score.

PubMed Disclaimer

Figures

FIGURE 1
FIGURE 1
The flowchart of this study.
FIGURE 2
FIGURE 2
The landscape of genetic variation of 5mC regulators in HCC Panel. (A) Data on the somatic mutation and copy number variations of 21 5mC regulators. (B) Waterfall plot of somatic mutation. (C) Location of CNV alteration of 5mC regulators on chromosomes. (D) Correlation between 21 5mC regulators in HCC. (E) The results of differentially expressed analysis from 5mC regulators. In panel (E), *p < 0.05, **p < 0.01, ***p < 0.001.
FIGURE 3
FIGURE 3
Establishment, verification, and heatmap of 5mC related molecular subtypes. Panels (A,B) show the most appropriate value for cluster. (C) Results of PCA. (D) Survival analysis. (E) Heatmap of subtypes.
FIGURE 4
FIGURE 4
Somatic mutations and TMB in different subtypes Panels (A–C) show the waterfall plots of clusters 1, 2, and 3, respectively. Panel (D) shows the TMB correlation analysis.
FIGURE 5
FIGURE 5
Relevant signaling pathways, immune cell infiltration, and immune checkpoints in subtypes. Panel (A) shows the results of gene set variation analysis. (B) Immune cell infiltration in the three subtypes. (C) Correlation between immune checkpoints and subtypes. In panel (B), *p < 0.05, **p < 0.01, ***p < 0.001.
FIGURE 6
FIGURE 6
5mC score construction. Panels (A–C) show the volcano plots of the differentially expressed analysis. Panel (D) shows the Venn plot of three volcano plots. (E) Results of univariate Cox regression analysis. (F,G) Results of lasso regression analysis.
FIGURE 7
FIGURE 7
Exploration and validation of the 5mC score. Panel (A) shows the results of survival analysis, (B) shows the correlation analysis between 5mC score and subtypes, and (C) shows the result of gene set enrichment analysis.
FIGURE 8
FIGURE 8
Independent biomarker analysis and validation from the ICGC cohort. Panels (A,B) show the results of univariate and multivariate Cox regression analysis, respectively. (C) Survival analysis of the subtypes. (D) Survival analysis of 5mC score.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Agopian V. G., Harlander-Locke M., Zarrinpar A., Kaldas F. M., Farmer D. G., Yersiz H., et al. (2015). A novel prognostic nomogram accurately predicts hepatocellular carcinoma recurrence after liver transplantation: analysis of 865 consecutive liver transplant recipients. J. Am. Coll. Surg. 220 416–427. 10.1016/j.jamcollsurg.2014.12.025 - DOI - PubMed
    1. Aravalli R. N., Cressman E. N. K., Steer C. J. (2013). Cellular and molecular mechanisms of hepatocellular carcinoma: an update. Arch. Toxicol. 87 227–247. 10.1007/s00204-012-0931-2 - DOI - PubMed
    1. Burugu S., Dancsok A. R., Nielsen T. O. (2018). Emerging targets in cancer immunotherapy. Semin Cancer Biol. 52 39–52. 10.1016/j.semcancer.2017.10.001 - DOI - PubMed
    1. Callahan M. K., Postow M. A., Wolchok J. D. (2016). Targeting T cell co-receptors for cancer therapy. Immunity. 44 1069–1078. 10.1016/j.immuni.2016.04.023 - DOI - PubMed
    1. Cancer Genome Atlas Research Network (2017). Comprehensive and integrative genomic characterization of hepatocellular carcinoma. Cell 169 1327–1341. 10.1016/j.cell.2017.05.046 - DOI - PMC - PubMed