Identification of differentially expressed tumour-related genes regulated by UHRF1-driven DNA methylation

Abstract

Ubiquitin-like with PHD and RING finger domains 1 (UHRF1) is an epigenetic regulator that plays critical roles in tumours. However, the DNA methylation alteration patterns driven by UHRF1 and the related differentially expressed tumour-related genes remain unclear. In this study, a UHRF1-shRNA MCF-7 cell line was constructed, and whole-genome bisulfite sequencing and RNA sequencing were performed. The DNA methylation alteration landscape was elucidated, and DNA methylation-altered regions (DMRs) were found to be distributed in both gene bodies and adjacent regions. The DMRs were annotated and categorized into 488 hypermethylated/1696 hypomethylated promoters and 1149 hypermethylated/5501 hypomethylated gene bodies. Through an integrated analysis with the RNA sequencing data, 217 methylation-regulated upregulated genes and 288 downregulated genes were identified, and these genes were primarily enriched in nervous system development and cancer signalling pathways. Further analysis revealed 21 downregulated oncogenes and 15 upregulated TSGs. We also showed that UHRF1 silencing inhibited cell proliferation and migration and suppressed tumour growth in vivo. Our study suggested that UHRF1 and the oncogenes or TSGs it regulates might serve as biomarkers and targets for breast cancer treatment.

Figure 1

Characteristic analysis and comparison of methylated probes in UHRF1-shRNA and scramble MCF-7 cells. (A) qRT‒PCR analysis of the mRNA levels of UHRF1 in UHRF1-shRNA or scramble MCF-7 cells (left). The expression of UHRF1 was evaluated by immunoblotting with β-actin as the loading control (right). The data are shown as the mean ± SD from three independent experiments. *p < 0.05. (B) The proportion of CG context, CHG context and CHH context in total methylated C sites from UHRF1-shRNA or scramble MCF-7 cells. (C) Violin boxplot showing the methylation levels distribution in UHRF1-shRNA or scramble MCF-7 cells. (D) The CG context, CHG context and CHH context methylation levels in gene region distribution are shown. (E) Cluster heatmap of CG context. (F) The CHG context DMR distribution in the gene region. (G) The DMRs distribution on chromosomes and significantly different are shown. TE, repeat element.

Figure 1

Characteristic analysis and comparison of methylated probes in UHRF1-shRNA and scramble MCF-7 cells. (A) qRT‒PCR analysis of the mRNA levels of UHRF1 in UHRF1-shRNA or scramble MCF-7 cells (left). The expression of UHRF1 was evaluated by immunoblotting with β-actin as the loading control (right). The data are shown as the mean ± SD from three independent experiments. *p < 0.05. (B) The proportion of CG context, CHG context and CHH context in total methylated C sites from UHRF1-shRNA or scramble MCF-7 cells. (C) Violin boxplot showing the methylation levels distribution in UHRF1-shRNA or scramble MCF-7 cells. (D) The CG context, CHG context and CHH context methylation levels in gene region distribution are shown. (E) Cluster heatmap of CG context. (F) The CHG context DMR distribution in the gene region. (G) The DMRs distribution on chromosomes and significantly different are shown. TE, repeat element.

Figure 2

Functional enrichment analysis of the DMGs. (A) Pie chart showing the DMR quantity and proportion of CG context, CHG context and CHH context that were identified. (B) The DMRs were annotated and anchored into promoters and gene bodies, including 488 and 1696 genes that were hypermethylated and hypomethylated in promoters and 1149 and 5501 genes that were hypermethylated and hypomethylated in gene bodies. (C–F) BP, CC, MF, and KEGG pathway enrichment analysis of the hypomethylated promoter or hypomethylated gene bodies. GO: Gene Ontology, BP: biological process, MF: molecular function, CC: cell component, KEGG: Kyoto Encyclopedia of Genes and Genomes. Significantly enriched biological processes were ranked by p value, and the top ten Gene Ontology terms are shown. For KEGG terms, the top twenty-five terms are shown. The size of the dots represents the number of enriched genes. The larger the dot is, the more genes are enriched.

Figure 3

Functional enrichment and protein–protein interaction network analysis of the methylation-regulated differentially expressed genes. (A) an intersection analysis of DEGs and DMGs revealed 139 hypo-promoter or 78 hypermethylated gene body/upregulated genes, and 271 hypomethylated gene body or 17 hypermethylated promoter/downregulated genes. (B–E) BP, CC, MF, and KEGG pathway enrichment analysis of the methylation-regulated differentially expressed genes. (F) and (G) PPI network analysis of the methylation-regulated upregulated genes (F) and downregulated genes, and the networks were visualized by Cytoscape software. The hub genes are shown in red.

Figure 4

Identification of the oncogenes and TSGs. (A) Twenty-one overlapping genes of the methylation-regulated downregulated genes and oncogenes (left), and fifteen overlapping genes of the methylation-regulated upregulated genes and TSGs (right) are shown. (B) Heatmap showing scaled expression of the oncogenes and TSGs. (C) The PPI network of oncogenes and TSGs. The red spots represent the oncogenes, and the green spots represent the TSGs. (D) and (E) KEGG enrichment analysis (D) and GAD disease analysis (E) of the oncogenes and TSGs. (F) qRT‒PCR validation of the oncogenes and TSGs. Red represents the oncogenes, and green represents the TSGs. (G) A visual summary shows the genetic alteration of the TSGs in breast cancer patients using data from the cBio Cancer Genomics Portal (http://www.cbioportal.org/).

Figure 4

Identification of the oncogenes and TSGs. (A) Twenty-one overlapping genes of the methylation-regulated downregulated genes and oncogenes (left), and fifteen overlapping genes of the methylation-regulated upregulated genes and TSGs (right) are shown. (B) Heatmap showing scaled expression of the oncogenes and TSGs. (C) The PPI network of oncogenes and TSGs. The red spots represent the oncogenes, and the green spots represent the TSGs. (D) and (E) KEGG enrichment analysis (D) and GAD disease analysis (E) of the oncogenes and TSGs. (F) qRT‒PCR validation of the oncogenes and TSGs. Red represents the oncogenes, and green represents the TSGs. (G) A visual summary shows the genetic alteration of the TSGs in breast cancer patients using data from the cBio Cancer Genomics Portal (http://www.cbioportal.org/).

Figure 5

Knockdown of UHRF1 inhibits the motility and tumorigenic capacity of MCF-7 cells. (A) colony formation assay was performed to determine the colony formation ability of UHRF1-shRNA or scramble MCF-7 cells, and the relative clonogenicity was calculated. The data shown represent the mean ± SD of three independent experiments. ***p < 0.001, ANOVA. (B) A wound healing assay was performed to show the migration capacity of UHRF1-shRNA or scramble MCF-7 cells. Data are shown as the mean ± SD from three independent experiments. ***p < 0.001, ANOVA. (C) Tumour growth curve showing the growth of UHRF1-shRNA or scramble MCF-7 cell xenografts in vivo. (D) Pictures (left) and tumour weights (right) of UHRF1-shRNA or scramble MCF-7 cell xenografts. (E) Validation of the high UHRF1 level in Breast cancer on the Human Protein Atlas database(https://www.proteinatlas.org/). Data are shown as the mean ± SD from three independent experiments. ***p < 0.001, ANOVA.